ELSEVIER Molecular and Cellular Endocrinology 108 (1995) 143-148

1,2SDihydroxyvitamin D, reduces the number of a I-andrenergic receptors in FRTL-5 rat thyroid cells

Mikael Ahlstr6m@vc, Kid TijrnquisW, Christel Lamberg-Allardta~c~*

aCalcium Research Unit, Minerva Foundation Institute for Medical Research, Tukholmankatu 2, SF-00250 Helsinki, Finland bDepartment of Zoology, University of Helsinki, Helsinki, Finland

‘Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, Finland

Received 21 September 1994; accepted 16 December 1994

Abstract

Noradrenaline and ATP evokes a transient increase in the intracellular calcium concentration ([Ca2+]J in FRTL-5 cells. In a previous study, we showed that 1,25-dihydroxyvitamin-D3 (1,25(OH)2-D3) increases the ATP evoked changes in [Ca’+]i. In the present paper, we found that pre-incubating the cells with 10 nM 1,25(OH)2-D3 for 48 h did not affect the noradrenaline-evoked increase in [Ca’+]i. We subsequently examined if this could be due to an effect of 1,25(OH)2-DJ on al-adrenergic receptor number, or receptor affinity. Pretreatment with 10 nM 1,25(OH)2-D3 for 48 h decreased the binding of the at-adrenergic specific antagonist [3H]prazosin by 55%

(&ax for 1,25(OH)2-D3 treated = 27.6 + 5.0 fmoYmg protein, untreated = 61.7 * 5.4 fmoYmg protein). No effect of 1,25(OH)2-D3 on the affinity for [3H]prazosin was observed. The effect of 1 ,25(OH)2-D3 on the [3H]prazosin binding was both time- and dose-dependent and could first be seen after 8-12 h of 1,25(OH)2-D3 treatment, indicating a genomic effect. The effect of 1,25(OH)2-D3 could be abolished with the protein synthesis inhibitor cycloheximide. No effect on the [3H]prazosin binding could be seen after a 48 h pre- incubation with 100 nM of either 24,25_dihydroxyvitamin D3 and 25dihydroxyvitamin D3, indicating that the effect of 1,25(OH),-D3 was specific. The cellular CAMP concentration was decreased after 48 h treatment with 10 nM 1,25(OH)z-D3. When TSH was replaced with dibutyryl CAMP or forskolin the [3H]prazosin binding increased. 1,25(OH)*-D3 also reduced the dibutyryl CAMP and forskolin stimulated [3H]prazosin binding. In addition, 1,25(OH),D, reduced the [3H]prazosin binding in TSH deficient media. Our results suggest that 1,25(OH),-D3 reduces the number of al-adrenergic receptors via a dual mechanism: by reducing CAMP production and by affecting some mechanism(s) downstream from the production of CAMP.

Keywords: al- Adrenergic; 1,25-Dihydroxyvitamin-D3; Prazosin; Thyroid; FRTL-5 cells

1. Introduction

The FRTL-5 rat thyroid follicular cell line has been

widely used as a model in studies of the properties of thy- roid follicular cells (Ambesi-Impiombato, 1989). FRTL-5 cells depend on thyrotropin (TSH) for growth through a

mechanism that involves cyclic adenosine monophos-

phate (CAMP) as a second messenger. The iodide me- tabolism in thyroid follicular cells is regulated by both TSH and by al-adrenergic agents, such as noradrenaline

(NA). TSH affects both the uptake and efflux of iodide in thyroid cells. The TSH induced uptake of iodide is medi- ated by CAMP while both the TSH- and the NA-

- Corresponding author, Tel.: +358 0 4771005; Fax: +358 0 477

1025.

stimulated efflux of iodide is mediated by a Ca2+-

dependent pathway (Weiss et al., 1984a,b; Marcocci et

al., 1987). 1 ,25-dihydroxyvitamin-D3 (1,25(OH),-D3), the bio-

logically most active form of vitamin D, has been shown to affect important functions of a variety of tissues, in-

cluding endocrine cells (reviewed by Reichel et al., 1989; Walters, 1992). In earlier studies we have found func- tional receptors for 1,25(OH)2-D3 in FRTLJ rat thyroid

cells (Lamberg-Allardt et al., 1991). We also found that 1,25(OH)2-D3 reduces the TSH induced iodide uptake by reducing the iodide porter number (Lamberg-Allardt and Valtonen, 1992) . This was due to an effect on the CAMP signal as the CAMP induced iodide uptake was also re- duced. In addition, 1,25(OH)2-D3 has been found to affect several other important properties of the FRTLJ cell line,

0303-7207/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved

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144 M. Ahlstriim et al. /Molecular and Cellular Endocrinology 108 (1995) 143-148

such as cyclic AMP generation, [3H]thymidine incorpo- ration, and TSH receptor number (Berg et al., 1991, 1993,

1994; Ongphiphadhanakul et al., 1992). In FRTL-5 cells, addition of adenosine triphosphate

(ATP) as well as NA induces a transient increase in intra-

cellular Caz+ ([Ca2+]i). We have recently found that a 48 h

pre-incubation with 1 ,25(OH),-D3 enhances the ATP-

induced increase in [Ca2+]i (Tornquist et al., 1992). This

could at least in part be due to the decrease in CAMP in-

duced by 1,25(OH)2-D3 (Berg et al., 1991), as CAMP at-

tenuates the ATP-induced transient increase in [Ca2+]i (Tornquist and Ahlstrom 1993). It is believed that NA

acts in the same way as ATP on [Ca2+]i (Corda et al., 1985b; Tornquist, 1992). This led us to hypothesize that 1,25(OH)*-D3 could enhance the NA-induced increase in [Ca’+]i in the same way as it affects the [Ca2+li induced by

ATP. However, when FRTL-5 cells were pre-incubated

with l,25(OH)2-D3 for 48 h, there was no difference in

the NA-induced increase in [Ca2+]i. In the light of these

findings, we investigated the effect of 1,25(OH)2-D3 on

the affinity and number of the receptor that mediates the

biological effects of NA, namely the cr,-adrenergic recep-

tor. We used [3H]prazosin, a selective high-affinity radio- ligand for al-adrenergic receptors in this study. Our re-

sults show that 1,25(OH)?-D3 reduces the binding of [3H]- prazosin to FRTL-5 cells by reducing a,-adrenergic re-

ceptor number. A preliminary report of these findings has been presented (Ahlstrom and Lamberg-Allardt, 1994).

2. Materials and methods

2. I. Materials

Furanyl-5-[3H]prazosin (28 Ci/mmol) and iodinated CAMP (adenosine 3’,5’-cyclic phosphoric acid 2’-0-

succinyl 3-[*251]iodo TME, 2000 Ci/mmol) was purchas- ed from Amersham International (Buckinghamshire, UK).

Vitamin D metabolites were a gift from Roche O.Y.

(Espoo, Finland). Purified bTSH and CAMP antiserum were obtained from the National Institute of Diabetes and Digestive and Kidney Diseases. Forskolin, dibutyryl

CAMP, 3-isobutyl- 1-methylxanthine (IMX) and cyclohex-

imide were from Sigma Chemical Co. (St. Louis, MO, USA). Calf serum, culture medium and hormones for cell culture were from Gibco (Grand Island, NY, USA). Cul-

ture dishes were purchased from Falcon Plastics (Oxnard, CA, USA). Optiphase ‘HiSafe’ scintillation cocktail was from LKB Wallac (Turku, Finland). Fura 2/AM was ob-

tained from Molecular Probes (Eugene, OR, USA).

2.2. Culture techniques

FRTL-5 rat thyroid cells were a generous gift from Dr. Leonard D. Kohn (NIH, Bethesda, MD, USA). The cells were grown in Coon’s modified Ham’s F-12 medium, supplemented with 5% calf serum and a six hormone mixture composed of bTSH (0.3 mu/ml), insulin (IO~glml), hydrocortisone (3.6 ng/ml), transferrin (5 ,ug/

ml), glycyl-L-histidyl-L-lysine acetate (20 @ml), and somatostatin (10 ng/ml) (later referred to as 6H medium), containing 50 III/ml penicillin and 50 pg/ml streptomy-

cin. In some experiments bTSH was excluded from the media (later referred to as 5H medium). The cells were grown in a water saturated atmosphere of 5% C02/95%

air at 37°C on lOO-mm plastic dishes. The medium was

routinely changed twice a week. During the experiments

the medium was changed daily.

2 3 . . Measurement of [Ca2+].

Intracellular Ca2+ was ‘measured essentially as de- scribed earlier (Tornquist et al., 1992). Briefly, the me- dium was aspirated, and the cells were then harvested

with Hank’s balanced saline solution (HBSS buffer, con- taining 118 mM NaCl, 4.6 mM KCl, 10 mM D-glucose, 20 mM Hepes, 0.4 mM CaCl, pH 7.4) containing 0.1%

trypsin and 0.02% EDTA. After washing the cells three

times by pelleting, the cells were incubated with 1 PM Fura-2-AM for 30 min at 37°C. After the loading period

the cells were washed twice with HBSS, incubated for at

least 10 min at room temperature and then washed again.

The cells were added to a quartz cuvette, kept at 37°C and stirred throughout the experiment. Fluorescence was measured with a Hitachi F 200 fluorometer. The excita-

tion wavelengths were 340 and 380 nm, and the emission was measured at 510 nm. The signal was calibrated by addition of 1 mM digitonin to obtain F,,, and by chelat-

ing extracellular Ca2+ with 5 mM EGTA to obtain Fmtn. Tris-base was used to elevate pH above 8.3. [Ca2+]i WAS

calculated as described by Gryenkiewicz et al. (1985),

using a computer program designed for the fluorimeter

with a Kd value of 224 nM for Fura 2.

2.4. Binding studies

FRTL-5 cells were plated on 35 mm plastic dishes in

6H medium. Five days prior to an experiment the medium was changed to 5H medium. After this period the cells were grown for an additional 48 h in 6H medium contain-

ing 10 nM 1,25(OH)2-D3 or vehicle (ethanol). The bind-

ing of [3H]prazosin to FRTL-5 cells was assayed as de- scribed by Corda et al. (1985a). Briefly, the dishes were washed twice with 1 ml HBSS at 37°C and then placed in

1 ml of HBSS containing 400 pM [3H]prazosin (for Scat- chard plots 25-1600 PM). For evaluation of non-specific binding, 1OpM phentolamine was added to the mixture. After a 30 min incubation at 37°C the dishes were washed rapidly with 1 ml HBSS at 4°C. The dishes were then incubated with 1 ml ethanol at 20°C for 15 min to extract the [3H]prazosin from the cells. The extract was transferred to counting vials and the radioactivity was measured after mixing with 3 ml scintillation fluid.

2.5. Determination of cellular CAMP The culture conditions were essentially as described

above, with the exception that the medium contained

hf. Ahlstriim et al. I Molecular and Cellular Endocrinology 108 (1995) 143-148 145

0.25 mM IMX, and the cells were plated on 24-well plates instead of 35mm dishes. The experiments were

terminated by washing wells twice with 1 ml of cold HBSS followed by incubation with 1 ml of 2 mM HCl in ethanol at -18°C for 16 h to extract the CAMP. The ex-

tract was transferred to plastic tubes for evaporation in a

vacuum at 37°C. The CAMP concentration was then de- termined by radioimmunoassay as described by Frandsen

and Krishna (1976).

2.6. Data analysis The Kd and B,,, values were obtained by analysing the

binding data with a curve fitting computer program (Ligand@; Munson and Rodbard, 1980). Statistical calcu- lations were performed by Student’s t-test (for two means) and analysis of variance (for three or more means). The experimental results are expressed as means

f SD.

3. Results

3.1. 1,25(OH),-D, effects on the NA- and the ATP-evoked changes in [Ca2+/.

Pre-incubation’ of FRTL-5 cells with 10 nM

1,25(OH),-Ds for 48 h did not change the NA-evoked increase in [Ca’+]i. NA (lo-“ M) increased the [Ca2+li by 80 + 15 nM and 77 + 20 nM for the control cells and the

1 ,25(0H),D3-treated cells, respectively. The increase in

[Caz+]i evoked by 30pM ATP was 326 + 29.7 for control

cells and 502 f 43.4 for the l,25(0H)2-D3-treated cells

(P c 0.005, Fig. 1).

3.2. 1,25(OH)2-D3 effects on [3H]prazosin binding The binding of [3H]prazosin to FRTL-5 cells was

saturable to both control cells and to cells that had been

exposed to 10 nM 1,25(OH)2-Ds for 48 h (Fig. 2).

Scatchard plots of the binding data revealed that treating cells with 10 nM 1,25(OH),-D, reduced the [3H]prazosin

binding by 55%, compared to control cells (Fig. 3). The B was 61.7 + 5.4 fmol/mg protein for the untreated

ce!: and 27.6 f 5.0 fmol/mg protein for the 1,25(OH)2-

D3 treated cells.

The Scatchard plots were linear, suggesting only one binding site for [3H]prazosin. The affinity of the binding was not affected by 1,25(OH)2-Ds (Kd values were

0.06 + 0.02 nM and 0.05 f 0.02 nM for the 1 ,25(OH),-D3 treated and untreated cells, respectively). The effect of

1,25(OH),-D3 was dose dependent, and was significant (P c 0.05) at concentrations of 1 nM and higher (Fig. 4). The effect of 1,25(OH)2-D3 (P c 0.02) was observed after 8-12 h incubation with 10 nM 1,25(OH)2-D3 (Fig. 5). Incubating the cells with the vitamin D3 metabolites 24,25(OH)2-D3 and 25(OH)2-D3 at 100 nM concentrations for 48 h did not affect the binding of [3H]prazosin, thus confirming the specificity of 1,25(OH)2-D3 (Fig. 6). Adding 10,~M cycloheximide to the culture medium

200

1 NA a NA b

v v

Pi

u” 1000

Q 1 ATP C

ATP d

v v

Fig. 1. The effect of 1,25(OH)2-D3 on NA- and ATP-evoked changes in [Ca2’]i in FRTL-5 cells. The cells were treated with 10 nM 1,25(OH)2- D3 (b,d) or vehicle for 48 h (a,~). The cells were stimulated with IO- 4M NA (a,b) or 30pM ATP (cd) and the changes in [Ca’+]f were measured as a function of time. The horizontal line denotes 1 min.

abolished the effect of 1 ,25(OH)2-D3 on the 13H]prazosin

binding (Fig. 7).

TSH increases the number of at-adrenergic receptors

by a mechanism which is CAMP-mediated (Corda et al.,

1985a). We could confirm this as both 0.2 mM dibutyryl CAMP (Bu2 CAMP) and 30,uM forskolin in 5H medium increased the [3H]prazosin binding compared to cells in

5H alone (Fig. 8). The [sH]prazosin binding was de- creased when cells were incubated with 10 nM

1,25(OH),-Ds for 48 h in 5H medium containing 0.2 mM

60

I

250 500 750

c3 H) Prazosin (pmol/L)

Fig. 2. Saturation curves for the specific binding of [3H]prazosin to FRTL-5 cells maintained in 6H medium with (+), or without (Cl) IO nM 1,25(OH),-D3for 48 h.

146 M. Ahlstriim et al. I Molecular and Cellular Endocrinology 108 (1995) 143-148

IA

P

Kd = 0.06+/-0,02 nM Bmax q 61.7+1-5.4 fmoll mg protein

El

w I - I - , . , . , . ,

0 10 20 30 40 50 60 70

B (fmollmg protein)

085

0,4 -

Kd z O.OSt1~0.02 nM Bmax = 27.6tk5.0 fmol mg protein

w 0 10 20 30 40 50 60 70

B (fmollmg protein)

Fig. 3. Scatchard analysis of [3H]prazosin binding to FRTL-5 cells maintained in 6H medium without (upper panel) and with (lower panel) 10 nM 1,25(OH),-D3 for 48 h. The given Kd and B,, values (mean + SD) were obtained from three separate experiments by analys- ing the binding data with the Ligand@ computer program.

60 , I

0 0,Ol 0,l 1 10 20 100

nM 1,250

Fig. 4. Dose-response curve for the 1,25(OH)2-D3 (1,25D) effect on [‘H]prazosin binding to FRTL-5 cells. The cells were maintained in 6H medium with increasing amounts of 1,25(OH)2-D3 for 48 h. The bars give the mean * SD of triplicate dishes. ***PC 0.001, **PC 0.01).

65 -1

255 0 20 40 60 E 3

time(h)

Fig. 5. Time effect of 1,25(OH),-D3 on [3H]prazosin binding to FRTL 5 cells. The cells were maintained in 6H medium with (+). or without (0) IO,UM 1,25(OH)2-D3. The points give the mean f SD of triplicate dishes (P C 0.02, analysis of variance).

Bu2 CAMP or 30 PM forskolin (Fig. 8). This indicates that 1,25(OH)2-D3 affects the binding of [3H]prazosin by af- fecting the CAMP signal beyond the production of CAMP. 1,25(OH)2-D3 also reduced [3H]prazosin binding to cells incubated in TSH deficient medium (Fig. 8).

3.3. 1,2.5(OH)z-D3 effects on CAMP concentration Incubating FRTLJ cells for 48 h in 10 nM 1 ,25(OH)2-

D3 reduced the cellular CAMP concentration. This effect could be seen in cells grown in both 6H and 5H medium (Fig. 9). The CAMP concentration in 6H cells was 134 + 13 pmol/mg protein in the untreated, and 57 f 5 pmol/mg protein in the 1,25(OH)2-D3 treated cells (P c 0.001). The corresponding values for cells grown in 5H medium was 53 + 5 and 43 f 4 pmol/mg protein (P c 0.05).

70:

C 1,25D 25D 24,258

Fig. 6. The effect of different vitamin D metabolites on the

[3H]prazosin binding to FRTL-5 cells. The cells were maintained in 6H medium for 48 h containing 10 nM 1,25(OH)2-D3 (1,25D),lOfJ nM 24,25(OH)2-D3 (24,25D) or 100 nM 25(OH)2-D3 (25D). The ban give the mean f SD of triplicate dishes. **P C 0.01.

M. Ahlstriim et al. I Molecular and Cellular Endocrinology IO8 (1995) 143-148 147

60

40

20

0

24 48

time (h) with CHX

-I

Fig. 7. Effect of 10,uM cycloheximide (CHX) on the [3H]prazosin binding to FRTL-5 cells maintained in 6H medium with (open bars), or without (hatched bars) 1OnM 1,25(OH)z-D3. The bars give the mean f SD of triplicate dishes. ***P < 0.001; *P < 0.05.

4. Discussion

We have earlier shown that 1,25(OH),Ds enhances the ATP-evoked transient increase in [Ca’+]i, possibly by enhancing an influx of extracellular calcium (Tiirnquist et al., 1992). Noradrenaline, by binding to ai-adrenergic receptors, causes a transient increase in [Ca’+]i by similar mechanisms as ATP (Corda et al., 1985b; Tomquist 1992). Surprisingly, however, we were not able to show any effects of 1,25(OH)2-D3 on the noradrenaline-induced transient increase in [Ca2+]i. Therefore, we wondered whether these findings could be due to an involvement of 1,25(OH)2-D3 on the binding of noradrenaline to al- adrenergic receptors .

The pharmacology of the ai-adrenergic receptors, us- ing [3H]prazosin, has been studied in several tissues.

TSH -TSH BtJZcAMP Forskolin

Fig. 8. The effect of 10 nM 1,25(OH)2-D3 on the [3H]praxosin binding to FRTL-5 cells maintained in 6H medium and 5H medium and the effect of replacing TSH with dibutyryl CAMP (Bu2 CAMP) and forskolin. The cells were maintained in medium with (open bars) or without (hatched bars) 10 nM 1,25(OH)2-D3 (1,25D) for 48 h for each treatment. The bars give the mean f SD of triplicate dishes. ***p < 0.001, **P < 0.01.

+TSH -TSH

Fig. 9. The effect of 1,25(OH)2-D3 on cellular CAMP concentration. FRTL-5 cells were maintained in 6H or 5H medium containing 0.5 mM IMX, with (open bars) or without (hatched bars) 10 nm 1,25(OH)2-D3 for 48 h. The bars give the mean f SD of triplicate dishes. ***p < 0.001, *P < 0.05.

Some tissues seem to have multiple binding sites for [3H]- prazosin (Oshita et al., 1991; Mignot et al., 1989). How- ever, our results confirm earlier reports that FRTL-5 cells have only one binding site (Corda et al., 1985a). The subclass of the ai-adrenergic receptors in FRTL-5 cells has to our knowledge not been determined, although it has been suggested that FRTL cells mostly contains ais- adrenergic receptors (Shimura et al., 1990).

Earlier reports have shown that al-adrenergic recep- tors in FRTL-5 cells are markedly upregulated by TSH through a mechanism that involves elevation of cyclic AMP (Corda et al., 1985a). In the present study we have confirmed that TSH upregulates the number of al- adrenergic receptors. The TSH stimulated level of [3H]prazosin binding obtained by us was similar to that reported by Corda et al. (1985a), and for FRTL cells by Shimura et al. (1990). The basal binding of [3H]prazosin was, however, high compared to the data of Corda et al. (1985a). This could be due to variations in methods, al- though we cannot rule out that the properties of the cells used in these two studies are different, as the passage number of the FRTLJ cells used by us is not known.

In this paper we demonstrate that 1,25(OH)2-Ds re- duces both the basal, the TSH stimulated, the Bu2 CAMP and the forskolin stimulated level of al -adrenergic recep- tors in FRTLJ cells. The mechanism by which 1,25(OH)2-D3 decreases the [3H]prazosin binding is still obscure. Recent reports have shown that 1,25(OH),-D3 reduces the TSH stimulated cyclic AMP concentration in FRTL-5 cells by reducing the TSH receptor number and by increasing the level of the adenylate cyclase inhibitory G-protein Gi _ p (Berg et al., 1991, 1994). In the present study, we have confirmed that 1,25(OH),-D3 reduces the TSH stimulated cyclic AMP concentration. One reason for the reduction in [3H]prazosin binding by l,25(OH)2- D3 could thus be due to a decrease in the CAMP concen- tration. 1 ,25(OH)2-D3 also reduced the [3H]prazosin

148 h4. Ahlstriim et al. I Molecular and Cellular Endocrinology IO8 (1995) 143-148

binding when the at-adrenergic receptors had been upregulated by Bu2 CAMP and forskolin, which indicates that 1,25(OH),-Ds affects the CAMP signal after CAMP has been generated. This is in line with earlier observa- tions that 1 ,25(OH)*-D3 attenuates the TSH-stimulated iodide uptake by affecting the CAMP signal (Lamberg- Allardt et al., 1991; Berg et al 1993). Furthermore, the effect of 1,25(OH)*-D3 seems to be dependent on protein synthesis. This fact was supported by the time that was needed for the effect to appear and by experiments with the protein synthesis inhibitor cycloheximide.

We conclude, that in FRTL-5 cells, 1,25(OH),-Ds re- duces both the TSH stimulated and the basal number of al-adrenergic receptors through a mechanism that de- pends on protein synthesis. 1,25(OH),-Ds seems to reduce the at-adrenergic receptor number via a dual mechanism: by reducing CAMP production and by affecting some mechanism(s) downstream from the production of CAMP. The effect that 1 ,25(OH)z-D3 has on cri-adrenergic recep- tor number probably explains why no enhanced effect was observed on the NA-evoked increase in [Ca2+li.

Acknowledgements

This work was supported by grants from the Sigrid Juselius Foundation, the Ella and Georg Ehrnrooth Foun- dation and the Nordisk Insulinfond which are gratefully acknowledged.

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