©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Low Threshold Calcium Channels by Angiotensin II in Adrenal Glomerulosa Cells through Activation of Protein Kinase C (*)

Michel F. Rossier (§) , Hervé B. C. Aptel , Christophe P. Python (¶) , Muriel M. Burnay , Michel B. Vallotton , Alessandro M. Capponi

From the (1)Division of Endocrinology, University Hospital, CH-1211 Geneva 14, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In adrenal glomerulosa cells, low threshold voltage-activated (T-type) calcium channels play a crucial role in coupling physiological variations of extracellular potassium to aldosterone biosynthesis. Angiotensin II markedly reduced the activity of these channels by shifting their activation curve toward positive voltage values. This inhibition of the channels resulted in a marked decrease of the cytosolic free calcium concentration maintained by potassium. This effect was abolished by losartan, a specific antagonist of the angiotensin II AT receptor. Hormone action on T-type channels appeared to be mediated by protein kinase C because 1) it was mimicked by phorbol ester and diacylglycerol, and 2) it was significantly reduced by decreasing protein kinase C activity with specific inhibitors such as chelerythrine chloride or a pseudosubstrate of the enzyme, as well as by protein kinase C down-regulation. Similarly, protein kinase C activation reduced the cytosolic calcium response to potassium and the steroidogenic action of this agonist. Low threshold T-type calcium channels therefore appear as potential sites for the modulation of steroidogenesis by protein kinase C in adrenal glomerulosa cells.


INTRODUCTION

The regulation of voltage-operated calcium channels by intracellular messengers has been recognized as a way for hormones and neurotransmitters to exert their effect on cell function. The modulation of L-type (high threshold and dihydropyridine-sensitive) calcium channel activity by various protein kinases, including the cyclic AMP-dependent kinase and protein kinase C, as well as by GTP-binding proteins has been extensively documented(1, 2) . In contrast, the regulation of low threshold, T-type calcium channels is still poorly characterized.

In adrenal glomerulosa cells, both low and high threshold Ca channels have been described(3, 4) . The exquisite sensitivity of these cells to small variations of extracellular K concentrations, as well as their very negative membrane potential(5) , strongly suggest a role for T-type calcium channels in the steroidogenic response to this agonist(6) . The involvement of these channels in the sustained cytosolic free calcium ([Ca]) response to AngII()is more debated. Indeed, by closing some K conductances, AngII is expected to depolarize glomerulosa cells (5, 7, 8, 9) and, therefore, to activate T-type channels. However, the poor effect of nicardipine, a dihydropyridine blocking both L- and T-type channels, on the [Ca] response to AngII suggests that other Ca influx pathways are stimulated by the hormone(10) . In addition, AngII has been shown to antagonize the sustained [Ca] response induced by K (11, 12), suggesting a negative modulation of T channels by the hormone.

In the present study, we used the patch-clamp technique to demonstrate that AngII inhibits T-type Ca channels in bovine adrenal glomerulosa cells by shifting the channel activation curve toward more positive potential values. This inhibition of the channels is mediated by protein kinase C (PKC) and results in a marked reduction of the [Ca] response and aldosterone secretion induced by K.


MATERIALS AND METHODS

Percoll was obtained from Pharmacia Biotech Inc.. Tetrodotoxin, sodium ATP, sodium GTP, and nicardipine were purchased from Sigma, and CsBAPTA and fura-2 acetoxymethyl ester from Molecular Probes (Eugene, OR). 1-oleoyl-2-acetyl-sn-glycerol (OAG), sn-1,2-dioctanoylglycerol (DiC), phorbol 12-myristate 13-acetate (PMA), chelerythrine chloride, and thapsigargin were from LC Laboratories (Woburn, MA), AngII was from Bachem AG (Bubendorf, Switzerland), and protein kinase C pseudosubstrate peptide inhibitor was obtained from Peninsula Laboratories Inc. (Belmont, CA). Losartan (DuP753) was a generous gift from Dr. R. D. Smith, DuPont Merck Pharmaceuticals (Wilmington, DE).

Adrenal Glomerulosa Cell Isolation and Culture

Bovine adrenal glands were obtained from a local slaughterhouse, and glomerulosa cells were prepared by enzymatic dispersion, purified on a Percoll density gradient, and maintained in culture for 2-4 days, as described in detail elsewhere(4) .

Patch-Clamp Measurements

The activity of slowly deactivating (T-type) Ca channels was recorded under voltage clamp in the whole cell configuration of the patch-clamp technique, as described previously(4) . The bath solution contained (in mM): 117 tetraethylammonium chloride, 20 BaCl, 0.5 MgCl, 5 D-glucose, 32 sucrose, and 200 nM tetrodotoxin, and was buffered to pH 7.5 with 10 mM Hepes/CsOH. The patch pipette (3-6 megohm, Clark 150T, Reading, United Kingdom) contained (in mM): 85 CsCl, 10 tetrabutylammonium chloride, 6 MgCl, 5 sodium ATP, and 0.04 sodium GTP, and pH was buffered to 7.2 with 20 mM Hepes/CsOH. The pipette solution also contained 0.9 mM CaCl and 11 mM CsBAPTA in order to buffer free calcium below 50 nM. Agents were directly added to the bath or introduced in the patch pipette, as indicated in the legend of . Diacylglycerols (OAG and DiC) were stored in small aliquots at -20 °C, in chloroform and under N atmosphere to prevent oxidation by air. Before use, aliquots were dried under N, resuspended in experiment buffer and sonicated. The reference electrode was placed in a KCl solution linked to the bath with an agar bridge; the resulting liquid junction potential was smaller than 2 mV and has been neglected. The cell was voltage-clamped (Axopatch 1D, Axon Instruments Inc., Foster City, CA) at a holding potential of -90 mV and depolarized as indicated. Fairly round and small cells, with a diameter of approximately 20 µm and a membrane capacitance of 15.8 ± 5.6 picofarads (S.D., n = 50), were chosen in order to optimize the spatial voltage clamp. The Ba currents were filtered at 1 kHz and sampled at 6.2 kHz. Leak was subtracted either digitally after the experiment or automatically by a P/4 protocol (pclamp 5.5, Axon Instrument Inc.). In a few experiments, pipettes coated with Sylgard (Dow Corning, Seneffe, Belgium) instead of plain pipettes have been used for recording T-type currents and similar characteristics (kinetics of activation and deactivation, Vof activation and inactivation) have been observed under both conditions.

Cytosolic Free Calcium Measurements

For [Ca] determinations, freshly isolated glomerulosa cells were purified on a Percoll density gradient and resuspended in a Krebs-Ringer medium (4) at a concentration of 5 10 cell/ml. Cells were then incubated for 30 min at 37 °C in the presence of 2 µM fura-2 acetoxymethyl ester, washed, and immediately used for determination of the fura-2 fluorescence (excitation at 340/380 nm and emission at 500 nm) in a Jasco CAF-110 fluorometer (Hachioji City, Japan). The fluorescence signal was digitized (DaQSys 2.0, Sicmu, University of Geneva), and [Ca] was calibrated using the ratio values of emitted fluorescence (340 nm/380 nm) as described in Ref. 13.

Determination of Aldosterone Formation

Measurement of aldosterone production was performed as described elsewhere(10) . Glomerulosa cells, cultured for three days, were incubated at 37 °C in multiwell plates containing a Krebs-Ringer medium and various concentrations of potassium and PMA or DiC. At the end of the incubation period, the aldosterone content of the medium was determined by direct radioimmunoassay, using a commercially available kit (Diagnostic Products Corp., Los Angeles, CA). Cellular proteins were measured using the Coomassie Blue method of Bradford(14) .


RESULTS AND DISCUSSION

In adrenal glomerulosa cells, the low threshold, slowly deactivating (T-type) Ca channels appear perfectly suited for controlling Ca influx, a signal sufficient for the activation of steroidogenesis(15, 16) , in response to small depolarizations of the membrane. The analysis of slowly deactivating Ba tail currents, elicited upon cell repolarization after a short (20 ms) depolarizing pulse (Fig. 1, A and B), allowed us to discriminate between T- and L-type channels in these cells. Indeed, L channels have been shown to rapidly deactivate (in a few milliseconds) at -65 mV(6) , and T channels can be considered as exclusively responsible for the slowly decaying current(4) . The time constant of T channel deactivation at -65 mV, as assessed by single exponential fitting, starting 5 ms after cell repolarization, was 6.6 ± 0.3 ms (n = 61). The analysis of activation (voltage-dependent opening) and steady-state inactivation (lack of opening upon strong depolarization) characteristics of T channels (Fig. 1C) revealed the presence of a permissive ``window'' of voltage, in which activation and inactivation curves overlap. As a consequence, at these voltages, the channels are already partially activated but not yet completely inactivated. This window therefore determines the range of voltage over which a steady-state current can flow through T channels, and the relative amplitude of this current can be calculated (6) as a function of voltage using Ohm's law (Fig. 1D). The resting potential of glomerulosa cells has been estimated to be -79 mV(5) , a value that is close to the threshold of this current.


Figure 1: Electrophysiological properties of low threshold (T-type) calcium channels in bovine adrenal glomerulosa cells. A, slowly deactivating Ba currents, recorded in the whole cell configuration of the patch-clamp technique (see ``Materials and Methods''), were elicited upon repolarization after a short period of activation (20 ms) at various depolarizing potentials. Example of six superimposed tail currents evoked in a representative glomerulosa cell at -65 mV, after membrane depolarization to various voltages (-45 to +5 mV, steps of 10 mV) from a holding potential of -90 mV. The time constant of the slowly decaying current in this cell was 6.9 ± 0.1 ms. B, slowly deactivating Ba currents elicitable after steady-state inactivation at various potentials. Tail currents (time constant = 7.5 ± 0.2 ms) were similarly elicited (at -65 mV) in the same cell, but after steady-state inactivation of T channels for 10 s at various holding potentials (from -80 to -30 mV) and 20 ms of activation at +20 mV. C, activation and inactivation curves of the T-type channels. Voltage-dependent activation () and inactivation () of slowly deactivating currents were measured from traces similar to those presented in panelsA and B, respectively, and as described elsewhere (19). The maximal current, elicited at the time of cell repolarization, was determined by extrapolating the tail currents fitted to a single exponential. Current amplitudes were plotted as a function of test voltage, after fitting to Boltzman's equation and normalization to the maximum of the function (I). The potentials at which the ratio of currents I/I is 0.5 (V) were -23.5 and -50.3 mV for activation and inactivation, respectively. D, steady-state current flowing through T-type channels. The theoretical steady-state current (I) was determined as a function of voltage from the Ohm's equation (6): I= gmh(V- V), where g is the maximal barium conductance through T channels (when all channels are open) and arbitrarily chosen equal to 1.0, m and h are the fractions of open channels (I/I), calculated from Boltzman's equation in activation and inactivation experiments, respectively, and V is the reversal potential measured to be = +50 mV in this cell.



AngII, a physiological secretagogue of aldosterone, significantly shifted the activation curve of T channel toward positive voltage values, without affecting the inactivation curve (Fig. 2A and ). This resulted in a marked reduction of the size of the permissive voltage window and therefore of the amplitude of the steady-state current (Fig. 2B).


Figure 2: Inhibition of T-type channels by angiotensin II. A, angiotensin II effect on the T channel activation curve. Cultured glomerulosa cells were voltage-clamped as indicated in the legend of Fig. 1, and Ba currents were recorded before and 3 min after addition of 50 nM AngII to the bath. Each cell was independently analyzed; the parameters of Boltzman's function were determined, and the currents were normalized before being averaged. A, activation (, ) and inactivation (, ) curves were established, as described in the legend of Fig. 1, before (, ) or 3 min after exposure of the cell to 50 nM AngII (, ). Data are the mean ± S.E. from 13 cells obtained from 9 independent preparations. The mean Vfor inactivation was -53.5 mV before and -53.8 mV after hormone application, whereas the Vfor activation was shifted from -29.3 to -20.9 mV by the same treatment. B, inhibition of the steady-state current by angiotensin II. The theoretical steady-state currents, before and after hormone treatment, were determined as described in the legend of Fig. 1D.



This inhibitory action of AngII on T channels could also be demonstrated by measuring the decrease of the cytosolic free calcium concentration after stimulation of fura-2-loaded glomerulosa cells with K (Fig. 3A). After depletion of intracellular Ca pools and activation of the capacitative Ca influx with 400 nM thapsigargin (10), the addition of 12 mM KCl induced a marked and sustained rise in [Ca]. This maximal response to K was rapidly reduced upon stimulation with AngII (50 nM), and nicardipine (2 µM) completely blocked the residual T channels (10), which had remained unaffected by the hormone. The fact that [Ca] decreased below the basal level after nicardipine treatment suggests the presence of a basal T channel activity in some resting cells, a fact reported previously(10) . We have estimated that, at this concentration, AngII inhibited by 70% the nicardipine-sensitive [Ca] response to K (n = 7), a value in agreement with the inhibition predicted by the shift of the current activation curve (). The action of AngII was prevented by the presence of 10 µM losartan (DuP753), a specific antagonist of the AT receptor subtype (Fig. 3B).


Figure 3: Inhibition of the potassium-induced cytosolic calcium response by angiotensin II. A, fura-2-loaded cells were sequentially exposed to 400 nM thapsigargin, 12 mM KCl, 50 nM AngII, and 2 µM nicardipine. In traceB, 10 µM losartan was added 100 s before AngII. [Ca] was determined as described under ``Materials and Methods.'' The traces are representative of two independent experiments.



Since hormone-induced inhibition of Ca influx through T channels required activation of AT receptors but was not dependent upon Ca release from intracellular stores or activation of the capacitative influx, a possible role for PKC was investigated. Indeed, three exogenous activators of PKC, PMA (1 µM), OAG (50 µM), and, to a smaller extent, DiC (100 µM) mimicked hormone action by significantly shifting the activation curve (). Moreover, the effect of AngII was markedly decreased when PKC activity was partially reduced by exposure to chelerythrine chloride (1 µM), or when protein kinase C pseudosubstrate peptide inhibitor (17) was present in the patch pipette at a concentration of 10-100 µM. Protein kinase C down-regulation by a 24-h treatment with PMA (100 nM), which reduced enzyme activity by approximately 85%(18) , also partially prevented AngII action (). No shift of T channel activation curve larger than 2 mV was observed in control (unstimulated) cells or in cells exposed to 5 µM thapsigargin (19) or 25 µM forskolin (not shown), and no significant effect of the various treatments was observed on the inactivation characteristics of the channel (). The inhibition of the maximal steady-state current through T channels due to the shift of their activation curve was also calculated (). This inhibition, greater than 60% in the presence of AngII, OAG, or PMA, was significantly (p < 0.05) reduced to less than 40% after decreasing PKC activity.

Protein kinase C activation by DiC (100 µM) resulted in a marked reduction of the [Ca] maintained by potassium (Fig. 4B) with a kinetics similar to the inhibition induced by AngII (Fig. 4A). When added after diacylglycerol, AngII only reduced [Ca] minimally, a result suggesting that both agents act through the same pathway. The effect of DiC was concentration-dependent, 50% inhibition of the response to K being observed at approximately 100 µM (not shown). The low solubility of this agent in aqueous solutions prevented a use at concentrations above 300 µM.


Figure 4: Involvement of protein kinase C in angiotensin II-induced inhibition of the calcium response to potassium. A, fura-2-loaded cells were exposed to 200 nM thapsigargin, 9 mM KCl, 10 nM AngII, and 2 µM nicardipine. In B, addition of KCl was followed by addition of 100 µM DiC. The same experiment was repeated in three independent cell preparations, giving similar results.



The inhibition of the [Ca] response to K by DiC was not due to a ``desensitization'' of the cell to extracellular K. Fig. 5A shows the response to 3 mM step increases in [K] in control (untreated) cells and in cells pretreated for 5 min with 100 µM DiC. The response in treated cells was reduced at each K concentration (Fig. 5B), and the inhibition (44%) could not be overcome by increasing K. The EC for the [Ca] response was 5.6 ± 0.2 mM added K in control cells and 5.7 ± 0.2 mM in DiC-treated cells. This is in agreement with the predicted inhibition of the relative size of the steady-state current, without noticeable shift in the sensitivity to potential (Fig. 2B). Interestingly, other activators of PKC, such as OAG or PMA, were much less efficient in reducing the K-induced [Ca] response. This difference could possibly be explained by a less efficient activation of PKC in adrenal glomerulosa cells by these agents, as already suggested by others(20) .


Figure 5: Inhibition of the calcium response to potassium by dioctanoyl-glycerol. A, fura-2-loaded cells, either untreated (C) or exposed for 5 min to 100 µM DiC, were stimulated by stepwise increases in extracellular K concentration; basal [K]: 3 mM, step increase: 3 mM. B, mean increase in [Ca] induced by various increases in KCl concentrations, calculated from four independent experiments performed as in panelA. Data were fitted to a four-parameter logistic function.



As expected, PMA and DiC also markedly reduced aldosterone secretion activated by extracellular potassium (Fig. 6). PMA inhibited up to 80% of the steroidogenic response to 12 mM potassium in a concentration-dependent manner, with an IC of 42 nM (Fig. 6A). At 100 nM (Fig. 6B), this agent similarly reduced aldosterone production in response to increasing concentrations of potassium, without significantly affecting the sensitivity of steroidogenesis to the agonist. Although we cannot completely exclude a direct effect of PMA (and PKC) on the various steps of steroidogenesis, our data suggest that the main inhibitory action of PMA on aldosterone secretion is secondary to its effect on the Ca channel. Indeed, in a separate study, we have observed that when aldosterone production is activated by a rise of [Ca] induced by ionomycin, a selective ionophore, and not through opening of T channels, PMA does not affect steroid output(16) .


Figure 6: Protein kinase C-mediated inhibition of the steroidogenesis induced by potassium. A and C, concentration-dependent inhibition by PMA (A) and DiC (C). Aldosterone secretion induced by 12 mM KCl was inhibited by increasing concentrations of PMA or DiC; data of panelA (mean values ± S.E. from 3 independent experiments) were fitted to a sigmoidal four-parameter logistic function to determine an IC value of 42 nM. A maximal inhibition of 80% was achieved at 1 µM PMA. In C, the effect of DiC on unstimulated cells is also shown (n = 4). B and D, effect of 100 nM PMA (B) or 100 µM DiC (D) on the steroidogenic response to extracellular potassium. Data represent the mean values ± S.E. from 11 and 6 independent cell preparations for B and D, respectively. EC values were estimated after fitting the data to logistic functions and were not significantly different in treated and control cells.



DiC action on aldosterone secretion appeared somewhat more complex. Although a marked inhibition of aldosterone secretion was observed at 100 µM, a slight potentiation of the response to potassium was obtained at concentrations ranging between 1 and 30 µM (Fig. 6C). The relevance of this potentiation is currently under investigation in our laboratory, but it does not appear to be dependent upon T channel activity, as suggested by the lack of a positive effect of DiC on the [Ca] response (not shown). At 100 µM, DiC reduced steroidogenesis at each KCl concentration by approximately 40%. Like PMA, this agent did not affect the cell sensitivity to potassium.

The action of AngII on voltage-operated Ca channels in adrenal glomerulosa cells is quite controversial. A hormone-induced, PMA-insensitive, activation of L-type, but not of T-type, currents has been reported in Y1 cells, an adrenal cortical cell line(21) . However, an involvement of high threshold (L-type) channels in normal glomerulosa cells, whose resting potential is much more negative than that of Y1 cells, is questionable. Recently, McCarthy et al.(22) described a GTP-dependent activation of T-channels by AngII in bovine adrenal glomerulosa cells. In this study, AngII appeared to shift the channel activation curve toward more negative values without affecting the inactivation curve. Another group (23) showed evidence for a role for calmodulin-dependent protein kinase II in this effect of AngII. Although we have at the present time no explanation for the discrepancy between these results and our data, an inhibitory action of AngII on T channel is more in agreement with the well documented reduction by AngII of the [Ca] response to potassium(11, 12) . In non-differentiated NG 108-15 cells, AngII has been shown to reduce the T-type Ca current through the angiotensin AT receptor subtype, by a mechanism presumably involving a phosphotyrosine phosphatase(24) . In bovine adrenal glomerulosa cells, which almost exclusively express the AT subtype(25) , AngII appears to exert its action through the latter subtype (Fig. 3B) and PKC activation.

It might seem paradoxical that AngII, a potent agonist of aldosterone secretion, inhibits T-type Ca channels, whose activity is indispensable for the steroidogenic response to potassium. However, we recently demonstrated that the majority of the Ca entering the cell in response to AngII uses a different mechanism, namely the capacitative Ca influx pathway, which is regulated by intracellular Ca pools and independent of voltage-operated Ca channels(10) . In addition, the inhibitory effect of AngII on T channels, highlighted under voltage-clamp conditions, is partially balanced by its depolarizing action on glomerulosa cells(7) . Interestingly, as a consequence, the net hormonal effect on T channels appears to be different in resting or in potassium-stimulated cells,() but the physiological relevance of this observation remains to be determined. Finally, the steroidogenic action of AngII, in contrast to that of K, also involves Ca-independent mechanisms(11, 16) .

The inhibition of glomerulosa cell T-type Ca channels through a shift of their activation curve toward positive potentials is not the only modulatory mechanism of the activity of these channels. Indeed, atrial natriuretic peptide (ANP), an antagonist of aldosterone secretion, has been shown to reduce T-type current by selectively shifting the inactivation curve of the channel toward negative voltages (6). It is noteworthy that two different hormones inhibit the same channel by affecting distinct properties of this channel. Because ANP induces cGMP formation and therefore activates a cGMP-dependent kinase, one could speculate that the latter kinase is involved in the modulation of the channel by ANP and that various regulatory sites are present on the same channel.

In conclusion, the modulation of T-type Ca channels by AngII provides the hormone with a mechanism to finely control Ca entry and, therefore, steroidogenesis in adrenal glomerulosa cells. The demonstration of a complex hormonal regulation of this channel, by protein kinases, phosphatases, or G-proteins, should trigger further studies leading to the purification, cloning, and molecular characterization of such an important effector in glomerulosa cell function.

  
Table: Role of protein kinase C in the angiotensin II-induced shift of T-type calcium channel activation curve

The recording conditions were the same as those described in the legend of Fig. 2, but the shifts in the activation and inactivation curves (V) induced by AngII, PMA, OAG, or DiC were determined for each cell independently. These agents were directly added to the bath. The 5th group of cells was pretreated for 1 h at 37 °C in the presence of 1 µM chelerythrine chloride before being exposed to AngII (50 nM) in the continuous presence of chelerythrine. PKCI, the enzyme pseudo substrate peptide inhibitor (17), was introduced into the pipette solution at a concentration of 10-100 µM. The last group of cells was incubated for 24-32 h in the presence of 100 nM PMA before current recording. The steady-state current inhibition due to the shift of the activation curve observed in each experimental condition was also determined as in Fig. 2B, and expressed as percent inhibition of maximal current. Results are the mean values ± S.E., and the number of cells tested (n) is indicated. The statistical significance of the difference between V values of activation before and after treatment with AngII, PMA, OAG, or DiC was assessed by the Student's t test; NS, not significantly different from untreated cells.



FOOTNOTES

*
This work was supported in part by Swiss National Science Foundation Grants 32-39277.93 and 31-27727.89 and by the Ciba-Geigy Jubiläumsstiftung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a grant from the Prof. Max Clotta Foundation. To whom correspondence should be addressed. Tel.: 41-22-3729320; Fax: 41-22-3476486.

Recipient of a fellowship from Pfizer (Switzerland) and the E& Schmidheiny Foundation.

The abbreviations used are: AngII, angiotensin II; PKC, protein kinase C; OAG, 1-oleoyl-2-acetyl-sn-glycerol; DiC, sn-1,2-dioctanoylglycerol; PMA, phorbol 12-myristate 13-acetate; ANP, atrial natriuretic peptide.

M. F. Rossier, C. P. Python, M. B. Vallotton, and A. M. Capponi, unpublished data.


ACKNOWLEDGEMENTS

We are grateful to Drs. P. Q. Barrett and U. Lang for helpful discussions, to Drs. W. Schlegel and S. R. Rawlings for their comments concerning this manuscript, and to Dr. R. D. Smith (Du Pont Merck) for providing us with losartan (DuP753). We have also benefited from the excellent technical assistance of L. Bockhorn, G. Dorenter, and M. Lopez.


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