Upregulation P. COLSON, A. DUVOID,

of V1, vasopressin

receptors by glucocorticoids

JAVIER IBARONDO, G. DEVILLIERS, AND GILLES GUILLON

M. N. BALESTRE,

Centre National de la Recherche Scientifique-htitut National de la Santh et de la Recherche Mkdicale de Pharmacologic-Endocrinologie, 34094 Montpellier Cedex 5 France; and Departemento Bioquimica y Biologia Molecular, Facultad de Ciencias, Universidad de1 Pais Vasco, 48 080 Bilbao, Spain Colson, P., Javier Ibarondo, G. Devilliers, M. N. Balestre, A. Duvoid, and Gilles Guillon. Upregulation of V1, vasopressin receptors by glucocorticoids. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E1054-E1062, 1992.-WRK1 cells (a rat mammary tumor cell line) exhibit a vasopressinergic receptor of V1, subtype tightly coupled to phospholipase C. Addition of dexamethasone to the culture medium principally potentiated the vasopressin-sensitive accumulation of inositol phosphates and to a lesser extent the NaF-sensitive phospholipase C activity. On the opposite, such treatment was without effect on the basal level of intracellular inositol phosphates or on bradykinin- or serotonin-sensitive phosphoinositide metabolisms. Glucocorticoid receptors were probably involved in these actions since dexamethasone was found to be more potent than aldosterone or corticosterone. Dexamethasone treatment also increased the number of vasopressin binding sites without affecting its affinity for vasopressin or other specific vasopressin analogues. These results strongly suggest that dexamethasone principally acts at the vasopressin receptor level by affecting its synthesis and/or the translation of its mRNA and also affects the G protein that couples the V1, receptor to the phospholipase C. These results explain how glucocorticoids may regulate the transduction mechanisms involved in vasopressin actions on WRKl cells. They provide explanations for understanding the cross talk between adrenal steroids and hormones, which mobilize intracellular calcium. WRK1 cells; V1, vasopressin receptor; dexamethasone; phospholipase C GLUCOCORTICOID HORMONES exert a positive or negative influence on numerous cultured cell lines and tissues (see, for example, Ref. 9). Thus glucocorticoid hormones are known to influence differentiation of a variety of cells lines such as myoblast (1)) adipocyte (6)) pancreatic acinar (22), and A6 kidney epithelial cells (28) and to inhibit cell growth (13, 22, 23). Glucocorticoids also widely affect the signaling pathways of multiple hormones, for example, they activate (2,9,28,30) or inhibit (2) hormonal-sensitive adenylate cyclase activities in many cellular systems. Dexamethasone also affected both the basal (23) and the hormone-stimulated inositol lipid metabolism in many tissues (3, 7, 10, 11). The mechanisms underlining this cross talk between adrenal steroids and other hormone molecules are poorly understood. Probably, part of these actions were due to an effect of glucocorticoids on the synthesis of molecules involved in the hormonal actions such as hormonal receptors (9, 21) or coupling G proteins (31, 33). Previous studies of Rajerison and collaborators (29) have demonstrated, in rat kidney, that adrenalectomy reduced the vasopressin-sensitive adenylate cyclase activity and that injection of aldosterone to adrenalectomized rats partially restored this response. Similarly, dexamethasone was also shown to increase vasopressinsensitive adenylate cyclase in epithelial cells derived from the kidney of Xenopus Levis (28). Because dexamethasone increases the density of renal vasopressin

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0193~1849/92 $2.00 Copyright

receptors (V, subtype), it was concluded that adrenal steroids upregulated their synthesis. Alternatively, the Vlb vasopressin receptor subtype present exclusively in hypophysis (17) was shown to be insensitive to the glucocorticoid response. Neither addition of dexamethasone in culture medium nor adrenalectomy of rats modified the vasopressin-sensitive accumulation of inositol phosphate in cultured corticotroph cells (4) or in pituitary glands, respectively (34). To further document the cross talk between adrenal steroids and vasopressin, we have studied the influence of dexamethasone on the V1, vasopressin receptor (third subtype of the vasopressin receptor family). This study performed on WRKi cells exhibiting a high density of V1, vasopressin receptor (15) demonstrates that dexamethasone treatment increased the density of V1, vasopressin receptor and to a lesser extent favored the coupling between phospholipase C (PLC) and a specific G protein (14). MATERIALS

AND

METHODS

Chemicals. [3H-Ar$]vasopressin ( [3H]AVP, 70 Ci/mmol) was from New England Nuclear; unlabeled AVP and other AVP analogues were from Bachem, and myo-[3H]inositol was from Commissariat a 1’Energie Atomique. Aldosterone, dexamethasone, hydroxycortisone, cycloheximide, and actinomycin D were from sigma. D-myo-inositol 1-monophosphate [Ins( l)P], D-myo-inositol 1,4-bisphosphate [Ins(l,4)P,], D-myo-inositol 1,4,5trisphosphate [Ins(1,4,5)P,], D-myo-inositol 1,3,4-trisphosphate [Ins(1,3,4)P,], and D-myo-inositol 1,3,4,5tetrahisphosphate [Ins( 1,3,4,5)P4] were from Boehringer Mannheim. Ins(1,4,5)P, assay system was from Amersham. Other chemicals were of the highest grade available and were from the sources given previously (15, 20). Cell culture. WRK, cells were cultured as previously described (20). Cells were plated in 3.5-cm plastic dishes at a density of 5-7 x lo4 cells and were grown in a complete medium composed of minimal essential medium containing Earle’s salts, fetal calf serum (5% vol/vol), rat serum (2% vol/vol), glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 mg/ml). For experiments in which inositol phosphate accumulation was measured, 2 &i/ml myo-3H label was added in the culture medium. The medium was changed and replaced by the same one 2 days after seeding. Experiments were performed 3-4 days later. When the cells were incubated with cycloheximide or actinomycin D, the culture medium was changed again 16 h before the beginning of the experiment for one containing the appropriate drugs. When specified, WRKl cells were grown in a desteroidated culture medium. This medium has the same composition as the complete medium, but both rat and calf serum were desteroidated using the charcoal and dextran technique, as previously described (36). Dexamethasone or vehicle was added at different concentrations in the culture media for the last 24-48 h of culture. Control experiments demonstrated that such addition did not significantly affect the cell growth (see Table 4). Determination of [3H/AVP binding. Hormone binding was

0 1992 The American

Physiological

Society

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RECEPTORS

assayed on cell monolayers grown 5 days, as previously described (15). Briefly, the culture medium was removed and replaced by phosphate-buffered saline (PBS) containing LiCl (10 mM), bovine serum albumin (BSA), and glucose (1 mg/ml), and the cells were preincubated for 15 min at 37*C. The binding assays were initiated by replacing this medium with 800 ~1 of the same buffer prewarmed and containing the appropriate concentration of labeled hormone together with 1 PM tyrosine (total binding). Nonspecific bindings were determined in each condition by adding, in the incubation medium, 0.2 PM unlabeled AVP. At the end of the incubation period (6 min), the medium was rapidly removed, and 1 ml ice-cold PBS-was added. Cells were scraped off using a rubber policeman and were layered on a Millipore RAWP filter (2.5cm diameter, 1.2-pm pore size) moistened with PBS. Filters were washed three times with 3 ml PBS. Radioactivity retained on the filters was counted by liquid scintillation spectrometry, and specific binding (total - nonspecific) was determined. Similar experiments were performed on crude plasma membranes derived from WRK1 cells. Briefly, membranes were prepared as previously described (14) and were incubated for 35 min at 37°C in a medium containing 20-40 pg membranous protein, 10 mM tris(hydroxymethyl)aminomethane (Tris) HCl, pH 8.0, 1 mM MgCIB, 0.25 mM EDTA, 1 mg/ml BSA, 0.5 mg/ml bacitracin, 0.005% (wt/vol) soybean trypsin inhibitor, and 0.4 nM [3H]AVP without (total binding) or with 1 PM unlabeled AVP (nonspecific binding). The reaction was stopped by adding 3 ml ice-cold washing buffer (10 mM Tris HCl, pH 8.0,l mM MgCl,). Then this mixture was layered on a Millipore filter, as described above, and radioactivity retained on the filter was determined. Specific bindings were determined as for experiments performed on intact cells. Determination of inositol phosphate accumulation and inositol lipid turnover in intact WRK, cells. WRK1 cells were grown in the presenie of 2 &i/ml myo-[3H]inositol, as previously described (20). Before the experiment, the culture medium was removed and replaced by a culture medium deprived of myo[3H]inositol and serum. Cells were then incubated for 45 min at 37°C. This medium was replaced by a PBS medium containing 10 mM LiCl, 1 mg/ml BSA, and 1 mg/ml glucose, and the cells were preequilibrated for 15 min at 37°C. Then this medium was aspirated and replaced by an identical one prewarmed (37°C) containing the agonists to be tested. The basal and stimulated production of inositol phosphates were measured for a 6-min incubation period. After rapid removal of the incubation medium, 1 ml perchloric acid (5%) was added to stop the reaction, and the dishes were placed on ice. Total inositol phosphates [Ins(l)P + Ins(l,4)P, + Ins(l,4,5)P3] that accumulated were determined by filtration through Dowex 1 x -8 columns as previously published (20). Inositol lipids remaining in the WRK, membranes were extracted with chloroform and deacylated by alcaline hydrolysis (20). The water-soluble and 3H-labeled products of this deacylation [glycerophosphoinositol (GlyPtdIns), glycerophosphoinositol 4-phosphate, and glycerophosphoinositol 4,5-bisphosphate] were separated by anion exchange chromatography, as previously described by Creba et al. (8). High-pressure liquid chromatography separation of inositol phosphates from intact WRK, cells. The cells were cultured as described above, but 10 &i/ml myo-[3H]inositol were added to the culture medium. Prelabeled cells were incubated with the agent tested in the conditions described above. The reaction was stopped by aspirating the incubation medium and by adding 1 ml of solution containing 5 mM KH2P04, 10 mM EDTA, pH 6.3, 0~2 pg/ml Ins( l)P, Ins( l,4)P2, Ins( l,3,4)P3, Ins( l,4,5)P3, and Ins(l,3,4,5)P,. The extraction and the preparation of the samples were executed as previously described (25). High-pressure liquid chromatography (HPLC) separation of inositol phosphates was performed on a partisil lo-SAX (25 X 0.46 cm) l

BY GLUCOCORTICOIDS

El055

anion exchange column. After injection of the sample, the column was washed with distilled water for 10 min to remove any unbound 3H-labeled material. Then the elution of inositol phosphates was achieved by increasing the concentration of ammonium formate (adjusted to pH 3.7 with orthophosphoric acid) from 0 to 3 M. The flow rate was kept constant at 1.1 ml/min. Fractions were taken every 0.5 min. Aquasure II (1.5 ml; Amersham) was added to each fraction, and the radioactivity was counted. All results were corrected for quenching and expressed in disintegrations per minute. Determination of inositol phosphate accumulation on intact rat hepatocytes. Isolated parenchymal cells were prepared from male Sprague-Dawley rats as previously described (16). Cells were labeled 90 min at 37°C in Krebs-Henw !leit bicarbonate buffer supplemented with 10 mM glucose, :.5% BSA and 5 &i/ml myo-[3H]inositol. Then, cells were diluted to 2-3 million per ml and incubated in the presence or absence of dexamethasone for 3 h. After that, we measured the accumulation of inositol phosphates in the same buffer but deprived of myo[ 3H]inositol and dexamethasone, as previously described (16). Mass measurements of Ins(1,4,5)P,. WRKl cells were grown 5 days in a desteroidated medium. The culture medium was aspirated, and the cells were washed two times with 1 ml PBS medium and preincubated 15 min at 37°C in PBS plus BSA and glucose (1 mg/ml). AVP (1 PM final concn) or vehicle were then added in the medium, and the reaction was stopped 5 s later by adding perchloric acid (5%). The cellular extract containing the bulk of intracellular inositol phosphates were prepared as previously described for experiments using myo- [3H]inositol prelabeled WRK1 cells (25). The determination of the amounts of Ins(l,4,5)P, present in these extracts was performed using the Amersham Ins( l,4,5)P3 assay system kit. Electron microscopy. Cell monolayers were fixed in situ with 5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for 30 min at room temperature, washed in the same buffer, and postfixed with 1% 0~0~ in 0.1 M cacodylate buffer for 30 min at room temperature. After dehydration in graded series of ethanol solutions, the cells were embedded in Epon. Ultrathin sections were cut parallel to the culture plane, stained with uranyl acetate and lead citrate, and examined with a JEOL 2000Ex electron microscope. Data analysis. The data are presented as means t SE and are normalized to 1 x lo6 cells. Estimates of the concentration that produces the half-maximal response (ED& was obtained directly from the dose-response curves. Statistical analysis of the data was performed using the one-way analysis of variance (ANOVA). Homogeneity of variance was assessedby Bartlett’s test, and P values were obtained from Dunnett’s tables. RESULTS

Influence of dexamethusone treatment on inositol phw phate metabolism. As illustrated in Fig. 1, when WRKl cells were grown in the presence of 0.1 PM dexametha-

sone, the AVP-stimulated level of inositol phosphate accumulation was significantly enhanced (-60%). This effect was seen only after a 6-h incubation period and was maximal after 24 h. Thus we choose this preincubation condition for the other experiments. This dexamethasone effect was dose dependent and was found to be maximal at 0.1 PM (Fig. 2C). The EDsO for dexamethasone was 8 t 5 nM (3 distinct determinations). Dexamethasone did not significantly affect the basal level of inositol phosphate accumulation, whatever the concentration tested up to 1 PM. Similarly, aldosterone and hydroxycortisone produced similar effects (Fig. 2, A and B). As compared with dexamethasone, both produced the same maximal effect on AVP-stimulated

Downloaded from www.physiology.org/journal/ajpendo (036.084.117.182) on August 24, 2019.

UPREGULATION

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i

I

//

//

RECEPTORS

BY GLUCOCORTICOIDS

11

l

9

8 (-Log

AVP II

6

Time

(hours)

24

72

Fig. 1. Time course of dexamethasone effect on inositol phosphate metabolism. WRK1 cells cultured 3 days in complete medium containing myo-[3H]inositol were further incubated in same culture medium with or without 0.1 PM dexamethasone (control) for different periods. At end of treatment, cells were washed 2 times with phosphate-buffered saline (PBS) and incubated 15 min in same medium + 10 mM LiCl and 1 mg/ml bovine serum albumin (BSA). Arginine vasopressin (AVP, 0.2 PM; 0) or vehicle (0) was then added for additional 6-min period. Total inositol phosphates that accumulated were determined by-Dowex technique. Results are means k SE of triplicate determinations originating from single experiment representative of 2 and are expressed as disintegrations . min- l (dpm). lo6 cells- I. Note that dexamethasone treatment did not affect growth of WRK, cells. * Statistically different from control corresponding value (P < 0.05).

0 *

160

7

s

[Ml)

Fig. 3. Dexamethasone potentiation of AVP-sensitive accumulation of inositol phosphates. Myo- [ 3H] inositol prelabeled cells grown in desteroidated medium were incubated during last 48 h with 0.1 PM dexamethasone (0) or vehicle (o), as described in Fig. 1. At end of treatment, cells were washed two times with PBS buffer and preincubated 15 min at 37°C in PBS supplemented with 10 mM LiCl and 1 mg/ml BSA. Increasing amounts of AVP were added for additional period of 6 min, and total inositol phosphates that accumulated were determined (see MATERIALS AND METHODS). Results are means k SE of triplicate determinations originating from single experiment representative of 3 and are expressed as dpm/106 cells. Arrows indicate concentrations of AVP leading to half-maximal stimulation (ED&. * Statistically different from cells incubated without dexamethasone (P < 0.02).

N

r

’I

' Ins(l,4,5

Ins

I/

(1,4)P2

1

/

A--/ ’ I’

,/'r,ilg / /-

3

)P3 -/

/ 4’I I’ I’ ::

Ins (1,3,4,5) P4

*ns(l,&

‘I

IL’

___

I.

1

10

20

1

30 Time (mln)

L

I

I

40

50

60

Fig. 4. High-pressure liquid chromatography (HPLC) profiles of inositol phosphate isomers. Myo- [3H]inositol prelabeled cells (10 &i/ml) were incubated during last 48 h with 0.1 PM dexamethasone (---) or vehicle (-). Cells were washed and preincubated for 15 min at 37°C in PBS supplemented with 10 mM LiCl and 1 mg BSA. AVP (1 PM) was then added for additional 5 s. Reaction was stopped as previously described. Inositol phosphates that accumulated were separated on partisil SAX 10x column using discontinuous ammonium formate gradient (25). Radioactivity eluted from HPLC column every 30 s was measured, corrected for quenching, and plotted against elution time. Labeled inositol phosphate standards were run in parallel under same experimental procedures. Arrows indicate respective elution positions. See Table 1 for definitions.

L0,

Fig. 2. Pharmacology of steroid effects on inositol phosphate metabolism. Myo- [3H]inositol prelabeled cells were grown during last 24 h without (control) or with increasing amounts of hydrocortisone (A), aldosterone (B), or dexamethasone (C). Total inositol phosphates that accumulated in presence (0) or in absence (0) of 0.2 PM AVP were measured as described in Fig. 1. Arrows indicate concentrations of steroids leading to half-maximal effects. Results are means of 3 distinct experiments, each performed in duplicate. Basal accumulations are expressed as %basal control (100% = 10,000 k 2,000 dpm/106 cells). Results are for AVP-stimulated inositol phosphate accumulation, as %AVP-stimulated control (100% = 41,200 k 4,500 dpm/106 cells).

inositol phosphate accumulation. Yet, their EDS0 values were higher (185 t 50 and 85 t 30 nM, respectively, 3 distinct determinations) for aldosterone and hydroxycortisone. As shown on Fig. 3, dexamethasone treatment did not affect the concentration of AVP leading to half-maximal stimulation of inositol phosphate accumulation (EDSo = 1.4 t 0.3 and 1.3 t 0.3 nM, 5 distinct determinations for control and dexamethasone-treated cells, respectively).

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Table 1. Influence Incubation Time

5s

6 min

of dexamethasone

OF VASOPRESSIN

on accumulation

RECEPTORS

of different

inositol phosphate isomers Radioactivity,

Effector Added

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BY GLUCOCORTICOIDS

dpm

Dexamethasone

None

-

None

+

AVP

-

AVP

+

None

-

None

+

AVP

-

AVP

+

GlyPtdIns

Ins( l)P

Ins( 1,4)P2

Ins( 1,3,4)Pa

Ins{ 1,4,5)Pa

Ins( 1 ,3,4,5)P4

InsPs

InsPs

28,700 +3,000 5,900 k900 26,200 k2,OOO 5,700 t900 29,600 25,000 7,500 ~1,000 33,700 k7,OOO 6,000 t900

1,500 &150 1,500 t250 1,400 t40 1,500 t130 1,900 &200 1,700 t120 15,900 1,100 21,500 &3,700

670 t80 850 t170 3,800 tlO0 8,700 +l,lOO 1,000 t200 800 t50 13,700 2500 55,000 +3,000

160 t40 150 a0 200 t50 110 t10 200 t20 100 t20 1,000 t150 1,700 t50

330 k80 230 k30 1,400 t220 3,500 k400 550 t50 250 k40 380 *30 600 k60

630 t20 890 t150 550 tlO0 800 a00 620 t50 670 t40 1,000 tlO0 1,600 k350

4,400 t800 5,500 t600 4,100 t260 5,000 t600 4,400 2300 5,800 t500 4,000 k800 5,500 &1,300

205 t60 390 k80 220 t40 330 t60 350 t40 420 t40 300 t50 350 k30

Results are means t SE of triplicate determinations and are expressed in disintegrations/min (dpm) for each assay. GlyPtdIns, glycerophosphoinositol; InsP5, inositol pentakisphosphate; InsPs, inositol hexakisphosphate; + and -, with and without dexamethasone, respectively; AVP, arginine vasopressin. See text for other definitions. Myo-[3H]inositol prelabeled cells (10 &i/ml) were incubated 5 s or 6 min with or without 1 PM AVP as indicated in Fig. 4. Inositol phosphate isomers that accumulated were separated on partisyl SAX column (see Fig. 4). Radioactivity found under each peak of radioactivity was counted and corrected for quenching. We assumed that peaks of radioactivity eluted with same elution time as those of inositol phosphates corresponded.

It principally modified the maximal AVP response (potentiation of AVP-stimulated inositol phosphate accumulation due to dexamethasone = 60 t lo%, 5 distinct experiments). However, this increase greatly depends on the batch of serum used for the culture medium and varied from 30 to 120%. To analyze precisely the influence of steroids on AVPstimulated inositol phosphate accumulation, we performed HPLC experiments. Figure 4 illustrates typical HPLC profiles of inositol phosphates extracted from control or dexamethasone-treated cells incubated 5 s with 1 PM AVP. As seen in Fig. 4 and summarized in Table 1, a 48-h preincubation with 0.1 PM dexamethasone significantly enhanced the amount of the Ins(l,4,5)P3 and Ins( l,4)P2 that accumulated under a short incubation period with AVP. Increasing the duration of the AVP stimulation (15 min) led to a dexamethasone effect on Ins(l)P, Table 2. Influence of dexamethasone

treatment

on inositol lipid pools Treatment

Effector

GlyPtdIns

GlyPtdIns(4)P

GlyPtdIns( 1,4)P2

None 33,300+ 1,700 1,100+75 360233 None AVP 31,300+1,500 1,160+85 450t50 Dexamethasone 34,500+700 1,150+30 600t40 Dexamethasone AVP 29,OOOk1,000 950t65 480t30 Results are expressed in dpm/assay and are means t SE of quadruplicate determinations originating from single experiment representative of 2. No. of cells was found unaffected by dexamethasone treatment. GlyPtdIns(4)P, glycerophosphoinositol4phosphate; GlyPtdIns( 1,4)Pz, glycerophosphoinositol 1,4-bisphosphate. Myo- [ 3H]inositol prelabeled cells (0.5 x 10”) were incubated during last 24 h with 0.1 PM dexamethasone or with vehicle. At end of treatment, cells were incubated for 6 min at 37°C with or without 0.2 PM AVP. Reaction was stopped by adding 5 ml perchloric acid (PCA) 5% (see MATERIALS AND METHODS). Cells were scraped and centrifuged, and pellets were subjected to lipid extraction. These extracts were deacylated using NaOH (see MATERIALS AND METHODS), and glycerophosphoinositides deriving from different inositol linids were senarated on Dowex columns.

Ins( 1,3,4)P3, and Ins( 1,3,4,5)P4 accumulation. However, as already mentioned, dexamethasone did not affect the basal levels of the different species of inositol phosphate nor the levels of inositol phosphate isomers whose accumulations were insensitive to AVP (inositol pentakisphosphate, inositol hexakisphosphate). On the other hand, dexamethasone considerably reduced the basal level of GlyPtdIns (80% inhibition). To confirm these data, we also performed the mass measurement of Ins( 1,4,5)P3 accumulated in control or dexamethasone-treated cells. The WRKl cells were grown and treated with dexamethasone, as indicated in Table 1. The amounts of Ins( 1,4,5)P3 accumulated during a 5-s incubation period at 37OC were 10.0 t 0.2 (basal condition) and 40.0 t 1.2 pmol/106 cells (+l PM AVP) for control cells and 11.5 t 0.4 (basal condition) and 126.0 t 16 pmol/106 cells (+l PM AVP) for dexamethasonetreated cells. Dexamethasone treatment did not modify the bulk of inositol lipids present in cells incubated with or without Table 3. Specificity of dexamethasone

effect

Inositol Phosphates, dpm/106 cells Effecters Control

None

cells

3,100+200

Dexamethasone-treated cells

3,400&200

Serotonin (100 PM) 7,600+370 8,500+ 1,200 Bradykinin (1 PM) 6,800+130 6,300+100* AVP (1 ,uM) 13,400+700 23,600+ 1 ,OOO* Results are means t SE of quadruplicate determinations from single experiment (representative of 3 experiments). Myo- [ 3H] inositol prelabeled cells were incubated during last 24 h with 0.1 PM dexamethasone or with vehicle (control). Inositol phosphates that accumulated after 6-min incubation period with or without effecters were measured as indicated in MATERIALS AND METHODS. * Statistically different from control cells (P < 0.05).

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of phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5bisphosphate (PIP2) were found to be unaffected (see Table 2). As previously published (20)) bradykinin and serotonin also stimulate the inositol lipid metabolism of WRKl cells. Thus we have compared the effect of dexamethasone on these hormonal responses. As illustrated on Table 3, a 24-h preincubation with 0.1 PM dexamethasone slightly reduced the bradykinin response (20% inhibition) and did not significantly affect the serotonin response. WRKi cells were grown in the presence of calf and rat serum(see MATERIAL~ANDMETHODS). Thusendogenous steroids were present in the culture medium and probably reduced the effect of the exogenous steroid. To test this hypothesis, we cultured WRKi cells with control or partially desteroidated serum in the presence or absence of exogenous dexamethasone (see MATERIALS AND METHODS) and tested their AVP responses on inositol phosphate accumulation. As illustrated in Table 4, with desteroidated medium, the basal level of inositol phosphate accumulation was unaffected as compared with cells cultured in complete medium. On the contrary, the AVPstimulated accumulation of inositol phosphates was reduced by * 30% in desteroidated medium. Addition of 0.1 PM exogenous dexamethasone restored the AVP-stimulated inositol phosphate accumulation to values similar to those observed with cells grown in complete medium. Thus it appeared that the glucocorticoid effect is more pronounced when cells were cultured in desteroidated media. Yet, we preferred to grow WRKl cells in complete media since desteroidation affected the cell growth (Table 4) and the cell morphology (see Fig. 8). To further study the possible effect of dexamethasone on the coupling between the PLC and the G protein, we have also tested on intact cells the influence of dexamethasone on the NaF accumulation of inositol phosphates. In the presence of 10 PM AlC&, NaF stimulated in a time (results not shown)- and dose-dependent manner (Fig. 5) the accumulation of total inositol phosphates (EDSo = 30.6 t 1.4 mM, 4 distinct determinations). When similar experiments were performed with dexamethasone-treated cells, they were more responsible to low doses of NaF, as compared with control cells. The EDS0 of NaF was

t

AVP. The amount

Table 4. Influence of serum desteroidation Culture Serum

used

Conditions Dexamethasone

g

AVP Binding, site/cell

I

8

4-

Z lz30

rs % rOl0. z $

O-

I

I

1

I

0

10

20

30

4 40

IrJaFl (mM) Fig. 5. Dexamethasone potentiation of NaF-sensitive inositol phosphate accumulation. Myo- [ 3H] inositol prelabeled cells grown in desteroidated medium were incubated during last 48 h with (0) or without 0.1 PM dexamethasone (0). Cells were then washed 2 times with PBS and preincubated 15 min at 37°C in PBS LiCl medium. Increasing amounts of NaF + 10 PM A1C13were added to incubation medium, and reaction was allowed to proceed for 30 min. Total inositol phosphates that accumulated were measured by Dowex technique. Results are means t SE of 3 distinct experiments, each done in triplicate, and are expressed as dpm/106 cells. Arrows indicate EDs0 of NaF. * Statistically different from cells incubated without dexamethasone (P < 0.02).

slightly but significantly reduced (EDSo = 25.2 t 1.4 mM), and maximal inositol phosphate accumulation was weakly potentiated (146 t 15%, 4 distinct determinations).

on dexamethasone Specific

BY GLUCOCORTICOIDS

Influence of dexamethasone

treatment on AVP binding.

In these experiments, we analyzed the influence of a dexamethasone treatment on the binding parameters of the Vi, AVP receptor. As illustrated on Fig. 6, this receptor has an affinity of 3.4 t 0.9 nM for [3H]AVP (3.4 t 0.9 nM), and its maximal binding capacity was 22,800 t 2,000 sites/cell (3 distinct determinations). When similar binding experiments were performed on WRKi cells grown with 0.1 PM dexamethasone during the last 24 h, the AVP receptor density increased by -70% (38,300 t 5,000 sites/cell). Yet, the affinity of the receptor for [3H]AVP was unchanged [dissociation constant (&) = 3.6 t 1.0 nM, 3 distinct determinations]. These steroid effects persisted even if we measured the binding capacity on partially purified preparations of WRKi plasma membranes (see legend of Fig. 7). effects Total Inositol Phosphate Accumulation, dpm/W cells Basal condition

AVP

No. of Cells/Dish, million

Nondesteroidated 6,400+300 4,600klOO 19,200+500 0.81t0.11 Nondesteroidated + 10,700+ 1,500 4,400+300 26,100+1,500* 0.78kO.15 Desteroidated 6,200+500 4,6OOH300 13,200+450 0.27t0.07 Desteroidated + 21,200+4,800* 2,900+150 26,3OOk900* 0.27t0.05 Results are the means k SE of quadruplicate determinations from 5 distinct experiments for specific AVP binding and of 3 distinct experiments for total phosphate accumulation. WRKi cells were grown 3 days in complete culture medium and for additional 3-day period in 4 distinct conditions: in same complete medium with (+) or without (-) 0.1 PM dexamethasone, in medium prepared with desteroidated rat and calf serum at same final concentration with or without 0.1 PM dexamethasone. For experiments in which inositol phosphate accumulation was measured, myo-[3H]inositol (2 &i/ml) was added in different culture media. Inositol phosphate accumulations were determined by incubating my~-[~H]inositol prelabeled cells for 6 min at 37°C with or without 0.2 PM AVP. Total inositol phosphates that accumulated were separated by Dowex technique. Binding experiments were performed by incubating cells for 6 min at 37°C with 2 nM [3H]AVP in presence (nonspecific binding) or absence (total binding) of 0.5 PM unlabeled AVP. Specific binding was calculated as difference between total and nonspecific determinations. * Statistically different from cells grown in nondesteroidated medium (P < 0.05). Downloaded from www.physiology.org/journal/ajpendo (036.084.117.182) on August 24, 2019.

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1000 Bound

RECEPTORS

\

\

0

OF VASOPRESSIN

’

\

\

\

\

7,~~~ 10 9 8 7 6 Oexamethasone I-log [ M I)

2000 (dpml

Fig. 6. Influence of dexamethasone treatment on specific AVP binding. Left: WRK, cells were grown for 5 days, as described under MATERIALS AND METHODS, with (0) or without (0) 0.1 PM dexamethasone for last 24 h. AVP binding sites were measured by incubating cells for 6 min at 37°C with increasing amounts of tritiated AVP. Nonspecific binding determined in presence of 1 PM unlabeled AVP was substracted from total binding. Scatchard representations of specific binding are illustrated. Results are means of duplicate determinations of single experiment representative of 3 and are expressed as dpm specifically bound/ assay. Right: WRK, cells were grown during last 24 h with increasing amounts of dexamethasone. Specific AVP binding was measured by incubating cells for 6 min at 37OC with 2 nM [3H]AVP (total binding) or 2 nM [3H]AVP + 0.2 PM unlabeled AVP (nonspecific binding). Results are means of duplicate determinations of single experiment representative of 2 and are expressed as dpm specifically bound/assay. Ad’

’

’

’

’

’

’

BY

El059

GLUCOCORTICOIDS

The influence of cycloheximide, a potent inhibitor of protein synthesis, and actinomycin D, an inhibitor of RNA synthase, were also studied to determine the site of action of dexamethasone. Cycloheximide by itself slightly increased the specific binding of AVP on WRK1 cells grown in a culture medium deprived of endogenous dexamethasone. It completely prevented the increase of AVP binding sites induced by 0.1 PM dexamethasone (Table 5). Actinomycin D treatment by itself reduced the specific binding of AVP by -40%. Moreover, as already observed for cycloheximide, it completely suppressed the positive influence of dexamethasone on AVP binding sites (Table 5). Influence

of dexamethasone

treatment

on morphology.

As seen on Fig. 8, WRK1 cells grown in control medium showed numerous mitochondria, some lysosomes, vacuoles, and some thin bundles of microfilaments under the plasmic membranes. They were not well differentiated since only few isolated intermediate filaments were visible in the cytoplasm. Cells grown in a partially desteroidated medium (Fig. 8B) exhibited important pinocytotic structures in the cytoplasm. This would probably signify that cells were suffering. When dexamethasone was added to control medium, no significant morphological modifications could be observed (Fig. 8C). On the contrary, when dexamethasone was added to desteroidated culture medium, the cells became more differentiated than controls. Numerous bundles of intermediate filaments, as well as isolated filaments, were scattered throughout the cytoplasm. DISCUSSION

W’

11

’

10

’

9

’

8

’

’

’

7 6 5 Unlabeled analogues

W’

’

11 10 (-Log [MI)

’

9

’

8

’

7

’

6

t

5

Fig. 7. Pharmacology of AVP binding sites in control and dexamethasone-treated cells. WRKi cells grown in desteroidated medium were incubated during last 48 h with 0.1 PM dexamethasone (B) or with vehicle (A). Crude plasma membranes were prepared from dexamethasone-treated or control cells and were incubated with 0.4 nM [3H]AVP with or without increasing amounts of unlabeled AVP analogues (v, AVP; 0, 1-deamino-[8-D-arginine]vasopressin; q , oxytocin; A, [d(CH,),,Tyr(Me)2,Argslvasopressin}. Specific bindings were determined in each condition and are expressed as %specific binding measured in absence of unlabeled analogue (100% = 118 t 6 and 292 t 3 fmol [3H]AVP specifically bound/mg protein for membrane derived from control or dexamethasone-treated cells, respectively). Results are means of duplicate determinations originating from single experiment representative of 2.

This dexamethasone effect on specific AVP binding was dose dependent (Fig. 6). The EDS0 of dexamethasone was -10 nM, a value similar to that found for the potentiation of the AVP-stimulated inositol phosphate accumulation (Fig. 2). Pharmacological studies were also performed on WRKl plasma membrane preparations derived from control or dexamethasone-treated cells to assess whether dexamethasone treatment modifies the binding properties of the AVP receptor. Data presented in Fig. 7 indicated that the AVP receptors present on the two membrane preparations exhibited the same pattern of recognition for a series of AVP analogues selected on the basis of their selectivity for AVP receptor subtypes (see, for example, Ref. 17).

Dexamethasone was able to potentiate the AVP-stimulated inositol phosphate accumulation in WRKl cells in a time- and dose-dependent manner (Figs. 1 and 2). Under the same experimental procedures, dexamethasone did not modify the basal level of inositol phosphate accumulation nor the pools of inositol lipid, suggesting that neither the amounts nor the catalytic properties of the PLC or enzymes involved in the inositol lipid turnover were affected (Fig. 2 and Table 2). Dexamethasone most probably increases the activity of an AVP-sensitive PIP2PLC since, using two different techniques [measurement Table 5. Influence of cycloheximide and actinomycin D treatments on increase of specific AVP binding sites induced by dexamethasone Treatment

[ 3H] AVP

Specific %control

Binding,

None lOOt17 Dexamethasone 233k34 Actinomycin D 57k3 Actinomycin D + dexamethasone 37k6 Cycloheximide 135k18 Cycloheximide + dexamethasone 103t17 Results are expressed as %control and are means k SE of 4 distinct experiments, each done in triplicate (100% = 8,800 t 1,500 sites/cell). WRK1 cells were grown in complete culture medium. After 2 days, medium was discarded and replaced by desteroidated one. Before beginning of experiment (16 h) performed at day 5, different drugs were added alone or in combination (3 pg/ml actinomycin D, 5 pg/ml cycloheximide, 0.1 PM dexamethasone). Control cells were grown in parallel without any treatment. Binding experiments were performed as indicated in Table 4.

Downloaded from www.physiology.org/journal/ajpendo (036.084.117.182) on August 24, 2019.

Fig. 8. Electron microscopy of WRKr cells. WRKr cells were grown for 5 days in 4 different culture media (see Table 4), fixed, stained, and examined, as described under MATERIALS AND METHODS. A: complete medium (te); B: desteroidated medium (dest); C, complete medium + 100 nM dexamethasone (dexa); D: desteroidated medium + 100 nM dexamethasone (desta + dexa). Arrows Downloaded indicate intermediate filaments. from www.physiology.org/journal/ajpendo (036.084.117.182) on August 24, 2019.

UPREGULATION

OF VASOPRESSIN

Table 6. Influence

of dexamethasone treatment hepatocyte phosphoinositol lipid metabolism Pretreatment

Assay

Total Inositol Phosphates Accumulated, cpm/LDH U

+ AVP (0.1 PM)

1,930+80 18,740+290 1,920&140

Control Dexamethasone

on rat

+ AVP (0.1 PM) 20,920+450* Results are means k SE of quadruplicate determinations from 1 experiment (representative of 3) expressed as dpm/lactate dehydrogenase (LDH) U, as previously described (16). Myo- [3H]inositol prelabeled rat hepatocytes (2-3 million/ml) were incubated 3 h at 37°C with 1 PM dexamethasone or with vehicle. At end of treatment, cells were washed with incubation medium (see MATERIALS AND METHODS) and preincubated for 15 min in same medium supplemented with 10 mM LiCl. Cells (1-1.5 million/ml, 1 ml/assay) were further incubated at 37°C in presence or absence of 0.1 PM AVP for 15 min. Reaction was stopped by adding to cell suspensions 0.3 ml 10% ice-cold PCA. Inositol phosphates were extracted and separated (see MATERIALS AND METHODS). * Statistically different from corresponding values (0.02 < P < 0.05).

of [3H]Ins( 1,4,5)P3 or of total unlabeled Ins( 1,4,5)P3 by radioreceptor assay], we show that dexamethasone potentiates the AVP-sensitive accumulation of this inositol trisphosphate isomer (Table 1). This glucocorticoid treatment may either modify the number of AVP receptors and/or alter the coupling between the V1, receptor and the PIP,-PLC. Many arguments favored the following first hypothesis. 1) Dexamethasone increased the maximal hormonal accumulation of inositol phosphate without affecting ED 5. of AVP (Fig. 3). 2) Dexamethasone treatment increased the density of V1, receptor and did not affect its affinity for a series of AVP analogues (Figs. 6 and 7). 3) The EDso of dexamethasone on AVP-sensitive phosphoinositide metabolism and on AVP specific binding were similar (Figs. 2 and 6). 4) Finally, when binding and inositol phosphate experiments were performed with the same batch of culture medium, dexamethasone similarly affected the increase of AVP binding sites and the AVP-stimulated accumulation of inositol phosphates (Table 4). Yet, we cannot underestimate some possible effects at the coupling level. Because the NaF-sensitive PLC activity was slightly affected by dexamethasone treatment (Fig. 5), we can assume that steroid treatment either weakly increased the amounts of the coupling G protein and/or modified its affinity for the PLC. This effect of steroid seems to be rather specific. Among the three hormones or the neurotransmitter tested in this study, only the action of AVP on phosphoinositide metabolism was upregulated by dexamethasone (Table 3). Moreover, these effects were not restricted to WRKl cells. Similar experiments performed on freshly isolated rat hepatocytes also indicate that a dexamethasone treatment (3 h at 37°C with 1 PM dexamethasone) increases the AVP-sensitive accumulation of inositol phosphate by - 13% (Table 6). This potentiation effect is similar to those obtained on WRK1 cells after the same time of treatment (Fig. 1). The phenomenon observed probably involved the interaction of dexamethasone with a specific glucocorticoid receptor since 1) dexamethasone was more active than

RECEPTORS

BY GLUCOCORTICOIDS

El061

hydrocortisone and aldosterone (Fig. 2) and 2) the active concentrations of dexamethasone (EDso -10 mM) were compatible with the affinity of the glucocorticoid receptors for this steroid (& varied from 6 to 20 nM according to the tissues tested; see Refs. 26, 35). Moreover, as described on other systems (2, 26), a prolonged exposure with steroids is necessary to observe the maximal effect of dexamethasone (Fig. 1). This probably represents the time necessary for glucocorticoids to act at the nuclear level and to stimulate the transcription and/or the transduction of the gene encoding for the AVP receptor. Such an assumption is corroborated by the fact that both cycloheximide, a potent inhibitor of protein synthesis, and actinomycin D, an inhibitor of RNA polymerase, completely suppressed the effect of dexamethasone (Table 5). In summary, we demonstrated that dexamethasone potentiates the AVP response of WRKl cells by at least two distinct pathways: an increase of the V1, receptor density and a weak potentiation of the coupling between the G protein and the PLC. Similar observations have been described earlier in the case of hormonal receptors coupled to adenylate cyclase. Dexamethasone treatment 1) increases the density of hormonal receptors positively coupled to adenylate cyclase (see Ref. 9 for review), 2) affects the coupling between the hormonal receptor and the adenylate cyclase (9), 3) selectively modulates some hormonal responses (2), and 4) alters the activity of enzymes involved in second-messenger degradation. By contrast, few studies have been devoted to the effects of adrenal steroids on the action of hormones that regulate their target cells via the hydrolysis of phosphatidylinositol 4,5-bisphosphate and the calcium mobilization. Our study and those of other groups show that, according to the receptors considered, glucocorticoid treatment may supersensitize or inhibit or is without effect on the hormonal stimulation of PLC activity (3, 10, 11, 23). This probably may account for the diversity of PLC enzymes being able to be activated by a hormonal receptor. Glucocorticoids differently affect the upregulation of AVP receptors. Yet, both the expression of the V2 and VI, receptor subtypes present in kidney and in many peripheral organs (liver or adrenal), respectively, are under the control of glucocorticoids (Refs. 28, 29 and this study). Alternatively, the Vlb receptor expressed exclusively in pituitary seems to be insensitive to adrenal steroids (4, 34). Such a difference with the V1, receptor confirms the specificity of the Vlb receptor previously established only on the basis of pharmacological studies (17). Studies are now in progress to elucidate the mechanism by which glucocorticoids upregulate the VI, AVP receptor. We are grateful to J. Bernard, M. Paolucci, M. Chalier, and A. L. Savage for typing and correcting the manuscript; M. Passama for drawing the figures; and Dr. S. Jard and D. Joubert for stimulating discussions. Address for reprint requests: G. Guillon, Centre CNRS-INSERM, de Pharmacologic-Endocrinologie, Rue de la Cardonille, 34094 Montpellier Cedex 5, France. Received 12 February 1992; accepted in final form 3 August 1992. REFERENCES 1.

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El062

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OF VASOPRESSIN

RECEPTORS

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