Adipocyte α2A-adrenoceptor is the only α2-adrenoceptor regulated by testosterone

ejp ELSEVIER

European Journal of Pharmacology Molecular Pharmacology Section 269 (1994) 95-103

molecular pharmacology ....

Adipocyte C 2A-adrenoceptor is the only c 2-adrenoceptor regulated by testosterone Anne Bouloumi6 a, Philippe Valet a Dani~le Daviaud a Herv6 Prats b Max Lafontan a,*, Jean-S6bastien Saulnier-Blache a a INSERM U317, Institut L. Bugnard, Universitd Paul Sabatier, CHU Rangueil, 3154 Toulouse Cedex, France b Laboratoire d'Endocrinologie Expdrimentale. Institut L. Bugnard, Facultd de Mddecine, CHU Rangueil, 31054 Toulouse Cedex, France

Received 15 December 1993; revised 7 April 1994; accepted 14 June 1994

Abstract

The effects of chronic administration of testosterone on a2-adrenoceptor expression in male hamsters were investigated in order to explore the selectivity of testosterone regulation towards the az-adrenoceptor subtypes. A homogeneous population of az-adrenoceptors was identified with [3H]RX821002 binding in adipocytes, colocytes and liver, whereas the c~2-adrenoceptor sites identified in kidney and brain were heterogeneous. Competition studies with az-adrenoceptor ligands characterized the presence of the azA-adrenoceptor subtype in adipocytes, colocytes, kidney and brain homogenates and of the azB-adrenoceptor subtype in kidney and liver. RNase protection assay with a selective hamster a2A-adrenoceptor cRNA probe confirmed the expression of a2A-adrenoceptor mRNA in adipocytes, colocytes, kidney and brain. Testosterone treatment did not modify the c~2-adrenoceptor densities whatever the subtype, except for the adipocyte azA-adrenoceptor, which was significantly increased. These results demonstrate that testosterone only up-regulates the adipocyte azA-adrenoceptor. Keywords: Adrenoceptor; Testosterone; Adipocyte; Colocyte; Kidney; Brain; Liver

1. I n t r o d u c t i o n

A large n u m b e r of tissues express a2-adrenoceptors and their activation leads to a wide spectrum of cellular responses. As regards their pharmacological and genetic characteristics, it is now well established that az-adrenoceptors compose a heterogeneous population. T h r e e different subtypes have been characterized: aZA, OZ2B and a z c , encoded by three distinct genes identified in man as a2-C10, a2-C2 and a2-C4, respectively (Bylund, 1992). Species-specific variations in the pharmacology of subtypes have been reported, especially for the O~2A-adrenoceptor between man and rodents (Link et al., 1992). The physiological relevance of such a heterogeneous population is still poorly defined but various observa-

* Corresponding author. Tel.: (33) 62 17 29 56; fax (33) 61 33 17

21. 0922-4106/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0922-4106(94)00101-4

tions suggest that a2-adrenoceptor subtypes may play distinct roles in the control of cell function. Studies describing the distribution of the ae-adrenoceptor subtypes revealed a tissue-specific profile. In the rat, tissues such as the brain contain the three subtypes (Nicholas et al., 1993), whereas two subtypes (a2A and a2B) were found in the kidney (Uhlen and Wilkberg, 1991) and a single subtype (a2A) was defined in colocytes (Paris et al., 1990). The factors involved in tissuedependent a2-adrenoceptor subtype expression are not known. Subtype differences towards agonist-induced desensitization have been described (Eason and Liggett, 1992), but there is no evidence that the a2-adrenoce ptor subtypes can undergo heterologous regulation. Androgens have been shown to be involved in the regulation of the a2-adrenoceptor expressed in hamster white adipose tissue depending on sex and sexual maturity (Saulnier-Blache et al., 1992). We investigated whether the testosterone effect was dependent on the a2-adrenoceptor subtype in the adult male hamster. The a 2-

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adrenoceptor subtypes expressed in isolated homogeneous populations of cells (adipocytes and colocytes) and in tissues composed of heterogeneous populations of cells (kidney, brain and liver) were characterized in male hamsters. A labelled selective az-adrenoceptor antagonist [3H]RX821002 (Saulnier-Blache et al., 1989) was used as well as a molecular probe specific for hamster ~2A-adrenoceptor transcripts. The presence of testosterone receptors in the studied cells and tissues was assessed by the use of the labelled specific androgen agonist [3H]R1881 (Bonne and Raynaud, 1975).

2. Materials and methods

2.1. Treatment of the animals The hamsters were maintained in accordance with the guidelines for care and use of laboratory animals (NIH Guide). All studies were performed on male Syrian hamsters (Mesocricetus auratus) of the same age (8 weeks) and weight (100 g), fed ad libitum and adapted to 20-22°C with free access to water. They were exposed to a long day photoperiod (16 h of light per day from 5 a.m to 9 p.m). Treatment consisted of one daily subcutaneous testosterone propionate injection (1 rag/animal in 0.1 ml of olive oil) over 7 days. After each experimental protocol, the animals were killed by decapitation and the different organs were dissected out. Adipocytes and colocytes were immediately isolated while the other tissues were frozen in liquid nitrogen.

2.2. Isolation of adipocytes and colocytes Adipocytes were isolated according to Rodbell's method (Rodbell, 1964) with minor modifications. The subcutaneous white adipose tissue was cut into small pieces and incubated for 30-40 rain at 37°C under vigourous shaking in Krebs Ringer bicarbonate buffer (pH 7.5) with 35 m g/ m l of bovine serum albumin, 6 mM glucose (KRBA buffer) and 1.6 m g/ m l of collagenase. After complete collagenase digestion, the adipocytes were separated from the stromal-vascular fraction by flotation and the packed cells were washed 3 times in KRBA buffer. The colonic epithelial cells were isolated from the intestinal mucosa as previously described by Laburthe et al. (1982). Intestine segments were flushed free of their contents with cold phosphate-buffered saline, everted and washed twice in the same solution. The following steps of the preparation were carried out at 4°C. Epithelial cells were isolated by scraping the everted intestine in a dispersing solution containing 2.5 mM EDTA (ethylene diamine tetraacetic acid) and 0.24 M NaC1 (pH 7.5). Cells were

collected by centrifugation and the pellets were washed once in phosphate-buffered saline.

2.3. Quantification of the a2-adrenoceptors Crude membranes were obtained after hypotonic lysis of the isolated cells (adipocytes and colocytes) or after homogenization of the tissues (kidney, brain and liver) in lysing medium with an Ultra-Turrax T 25 homogenizer (Janke and Kunkel) at 24000 rev/min, followed by centrifugation (800 g, 10 min, 25°C). The lysing medium was composed of 2.5 mM MgC12, 1 mM KHCO3, 2 mM Tris-HCl, 100 /xM EGTA (ethylene glycol-bis(/3-aminoethyl ether) N,N,N',N'-tetraacetic acid); pH 7.5 (35 mOsM) and the following protease inhibitors: 1 p,g/ml leupeptin, 0.1 mM benzamidine, 100/xM phenylmethyl-sulphonyl fluoride. Crude membranes pellets were obtained by centrifugation of the supernatants for the tissues or centrifugation of the homogenates for the isolated cells (40000 ×g , 10 min at 25°C). They were then resuspended in Tris-Mg 2+ buffer (50 mM Tris-HCl, 0.5 mM MgC12, pH 7.5) washed and collected by centrifugation (40000 × g, 10 min, 4°C). Binding experiments were conducted as previously described (Saulnier-Blache et al., 1989). Briefly, 100 Ixl of membrane suspension were incubated at 25°C in the presence of the radioligand in a 400 /xl final volume of Tris-Mg 2+ buffer. After a 45-min period of incubation, membrane bound radioligand was separated from free by rapid filtration through G F / C Whatman filters using a Skatron cell harvester. The filters were washed with cold Tris-Mg 2+ buffer, air-dried, transferred into vials and counted for radioactivity by liquid scintillation spectrometry. Specific binding was defined as the difference between total and non-specific binding determined in the presence of 10 - 4 M (-)-epinephrine. For saturation experiments the final concentration of [3H]RX821002 ranged from 0.25 to 20 nM. K D and Bm~ values were calculated by computer-assisted analysis of the data using the LIGAND program allowing statistical comparison between curve fitting to a one-site saturation model or to a two-site saturation model (McPherson, 1985). Inhibition studies were carried out at various radioligand concentrations as indicated (e.g. 2 nM [3H]RX821002 for adipocytes and colocytes, 6 nM for kidney, brain and liver) and increasing concentrations of drug competitors ranging from 1 pM to 1 raM. For inhibition studies with oxymetazoline, the binding medium was supplemented with 10 - 4 M GTP plus 100 mM NaCI. The inhibition constants (K~) were calculated from computer-assisted analysis of the data using the LIGAND program (McPherson, 1985). Specific binding was expressed as femtomoles of [3H]RX821002 bound and normalized to protein determined by the method of Lowry et al. (1951).

A. Bouloumid et al. / European Journal o f Pharmacology - Molecular Pharmacology Section 269 (1994) 95-103

2.4. Quantification of the androgen receptors

Rat RG 20 24

RATPYSLQVTLTLVCLAGLLMLFTVFGNVLVIIAVFTSRALKAPQ

Hamster

RATPYSLQETLTLVCLAGLLMLFTVFGNVLVIIAVFTSRALKAAQ

::::::::

Androgen receptors were quantified according to Roselli's method with some modifications (Roselli et al., 1991). Isolated adipocytes and colocytes were frozen directly in liquid nitrogen and kept at - 8 0 ° C until use. Thawed cells were homogenized in homogenization buffer (20 mM Tris, pH 7.4, 1.5 mM E D T A (ethylene diamine tetraacetic acid), 20 mM sodium molybdate, 12 mM monothioglycerol, 0.1% bacitracine) and centrifuged at 8 0 0 X g at 4°C for 15 min. The pellet containing the nuclei and the supernatant containing cytosols were separately collected. Whole tissues (kidney, brain and liver) were homogenized with an UltraTurrax T25 homogenizer (Janke and Kunkel) in 2 ml homogenization buffer. The homogenates were extracted with 0.4 M KC1 in homogenization buffer at 4°C and rapidly vortexed every 15 min for 1 h. The final suspensions were centrifuged at 15 000 × g for 60 min at 4°C to yield crude extracts. Aliquots (100 ml) of the extracts were incubated with increasing [3H]R1881 concentrations (0.5 to 10 riM) for Scatchard analysis of the data or routinely with 10 nM [3H]R1881 for 24 h at 4°C with or without a 200-fold exess of cold R1881 to determine total and non-specific binding respectively. Triamcinolone acetonide (10 mM) was added to the incubation medium to prevent binding of [3H]R1881 to progestin receptors. Bound and free [3H]R1881 were separated on Sephadex LH-20 columns. The radioactivity was measured in a liquid scintillation counter. Specific binding was expressed as femtomoles of [3H]R1881 bound and was then normalized to the protein content, as determined by the method of Lowry et al. (1951).

2.5. Polymerase chain reaction (PCR) cloning Hamster genomic D N A was amplified by PCR using the following oligonucleotide primers: primer h 5'-GGT CAA G C T T G A T G G CGC ACA G G T G C A C-3'; primer 2: 5'-JTI" T G A A T T CCA T G G GCT CCC T G C A G C CGG-3'). The primers were based on regions highly conserved ( + 1 to + 18 and + 364 to + 382) between the human az-C10 and the rat RG20 genes (Kobilka et al., 1987; Lanier et al., 1991). Endonuclease linkers were added at the 5' ends of the primers (NcoI in primer 2 and HindIII in primer 1) in order to facilitate cloning of amplified fragments. 1 /zg of hamster genomic D N A or 10 ng of purified vector containing the a2-C10 or RG20 genes were amplified with 1 /zg of each primer in 10 mM Tris-HC1 pH 9 / 5 0 mM KC1/1.5 mM MgC12/0.01% gelatine/0.1% Triton X100/250 mM of each dATP, dCTP, dGTP, d T T P / 5 units of Thermus aquaticus D N A polymerase (Taq D N A polymerase, Promega) in a final volume of 100 /zl. Incubations were carried out in a programmable

97

: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

:

NLFLVSLASADILVATLVIPFSLANEVMGYWYFGKVWCEIYLALD ::::::::::::::::::::::::::::::::::::::::::::: NLFLVSLASADILVATLVIPFSLANEVMGYWYFGKVWCEIYLALD VLFCTSSIVHLCAI

126

VLFCTSSIVHLCAI

Fig. 1. Alignment of the amino acid sequences deduced from the hamster amplified DNA fragment with rat RG20 (2). Identical amino acids are indicated by colons.

thermal controller (Biometra) and the amplification cycles were run as follows: 30 s of denaturation at 94°C followed by 30 cycles composed of 30 s denaturation at 94°C, 30 s of annealing at 60°C and 40 s of extension at 72°C. The reaction was stopped at 4°C after a final extension step of 4 min at 72°C. The amplified DNA fragment was purified by separation on a preparative 1.2% low-melting agarose gel, ligated into a pBluescript KS (_+) plasmid giving the ~2H390 vector. The nucleotide sequence of the amplified fragment was obtained by the method of Sanger (Sanger et al., 1977). Data base searches, deduced amino acid sequences and alignment of protein sequences were performed using G E N E P R O sequence analysis software (Riverside Scientific, Bainbridge Island, WA) (Fig. 1). The accession number assigned by GenBank data base is L28124. An equivalent sized fragment was also obtained from plasmids containing the whole coding sequence of the human a2-C10 gene (Kobilka et al., 1987) and the rat RG20 gene (Lanier et al., 1991). The percentage homology in peptide sequences with cloned a;-adrenoceptor subtypes was determined. The cloned hamster nucleotide sequence isolated from genomic D N A shared 96% homology with the rat RG20 sequence. Considering the deduced amino acid sequence, it exhibited more than 96% identity to a2a-adrenoce ptor subtypes (human ~2-C10 (Kobilka et al., 1987), rat R G 20 (Lanier et al., 1991), rat cA2-47 (Chalberg et al., 1990), porcine aaA-adrenoceptor gene (Guyer et al., 1990)), whereas the homology of sequence with o~2B-adrenoceptor subtypes (human c~2-C2 (Lomasney et al., 1990), rat R N G a 2 (Zeng et al., 1990)) was less than 84% and less than 73% with the a2c-adrenoce ptor subtypes (human ~z-C4 (Regan et al., 1988), rat RG10 (Lanier et al., 1991), rat pA2d (Voigt et al., 1991)). The cloned fragment of hamster oh-adrenoce ptor corresponds therefore to an ~2A-adrenoceptor subtype.

2.6. RNase-protection assay The plasmid a2H390 was linearized by BamHI and a labelled antisense R N A (cRNA) was transcribed in the presence of [32p]UTP using a T3 RNA polymerase. An unlabelled sense R N A was also transcribed from a

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HindlII-linearized a2H390 plasmid using a T7 RNA polymerase. In both cases, the DNA matrix was eliminated by exhaustive DNaseI-RNase-free digestion. Extraction of total RNA from cells or tissue was performed using the one-step guanidinium isothiocyanate/phenol/choroform extraction method (Chomczynski and Sacchi, 1987). The integrity of the RNA preparations was assessed by agarose gel electrophoresis and the concentration was measured by UV spectrophotometry. Hybridization was performed by mixing the labelled cRNA probe in 30 /xl of hybridization buffer (80% formamide, 0.4 M NaC1, 1 mM EDTA (ethylene diamine tetraacetic acid), 40 mM Pipes (piperazine-N,N'-bis[2-ethane-sulphonic acid]; 1,4piperazine-diethanesulphonic acid), pH 6.7) with the total RNA sample (Winter et al., 1985). The mixture was heated to 85°C for 5 min and immediately placed at 55°C for 14 h. Then, 0.5 ml RNase A (40 mg/ml) and RNase T1 (2 mg/ml) in 300 mM NaC1, 5 mM EDTA (ethylene diamine tetraacetic acid), 10 mM Tris-HC1, pH 7.5 were added. After 2 h at 37°C digestion was stopped by 5/xl protease K (10 mg/ml) for 15 rain at 37°C. The protected cRNA probe was extracted by phenol-chloroform and isopropanol-precipitated in the presence of 10/xg of tRNA. After a wash with 70% ethanol, the pellet was redissolved in 10 /~1 of loading buffer (97% formamide, 0.1% sodium dodecyl sulphate, 10 mM Tris-HCl, pH 7.0) and loaded on a 5%

acrylamide, 7 M urea gel. The gels were revealed by autoradiography at -80°C and the amounts of a2Aadrenoceptor RNA were determined by measuring the density of the specific band with a Biocom analyser. 2. Z Drugs and chemicals [3H]RX821002 (55 Ci/mmol) and [c~-32p]UTP (800 Ci/mmol) were obtained from Amersham International (Amersham, England). R1881 and [3H]R1881 (methyltrienolone) (86.5 Ci/mmol) were from New England Nuclear (Boston, MA). Prazosin was obtained from Pfizer (Belgium). Oxymetazoline, yohimbine, triamcinolone acetonide were from Sigma Chemical (St. Louis, MO). All other chemicals and biochemicals were reagent or molecular biology grade.

3. Results

3.1. Distribution of ae-adrenoceptors in tissues and cells 3.1.1. Saturation studies Studies were carried out to assess the binding properties of [3H]RX821002 on crude membranes of isolated cells (adipocytes and colocytes) and on total membrane extracts from brain, kidney and liver. In isolated cells, Scatchard analysis of saturation data (Scatchard, 1949) (Fig. 2A) showed that

10o

X

200,

80

u. 100,

40

?, m

20 0

0

100 20O 300 4~0

0

[3HI RX821002 Specific Bound 3o 20

o

io

LIVER

KIDNEY

~

• • • •

5

to

2oo

3oo

(fmol/mg protein)

50

10 m

0

lOO

75

2o

e

• E,

60

0

x

~,

15

0

~

• I0 20 [3H] RX821002 Specific Bound

25 0 o 20 (fmol/mgprotein)

lOO

200

Fig. 2. (A) Scatchard transformations of [3H]RX821002 binding data on isolated cells: adipocytes (black circles) and colocytes (open triangles). One representative experiment is shown. Mean values are given in Table 1. (B) Scatchard transformation of [3H}RX821002 binding data on kidney ([]), brain (11) and liver (©) obtained from one representative experiment. Mean K D values are given in Table 1.

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A. Bouloumid et aZ / European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 95-103

Table 1 Dissociation ( K D) and inhibition (K i) constants of [3H]RX821002 binding, expressed in nM, to membranes prepared from adipocytes, colocytes, liver, kidney and brain KD

Ki Yohimbine

Prazosin

High-affinity [3H]RX821002binding sites Adipocyte 0.50+0.06(3) 34_+10(3) Colocyte 0.58-+0.03(3) 48-+ 3 (3) Kidney 0.2 +0.1 (4) 6_+ 1 (5) Brain 0.3 _+0.1 (3) 23-+ 3 (3)

2261_+ 11 (3) 1632+305(3) 1247-+ 35 (4) 1871+291(3)

Low-affinity[3H]RX821002binding sites Liver 4.6 _+0.6 (5) 0.20_+0.04(4) Kidney 4.4 +1.3 (4) 0.20+ 0.06(5) Brain 8.2 _+1.2 (3) 0.40_+ 0.01(3)

182+ 75(3) 52_+ 18(4) 48_+ 22(3)

Data were analysed by computer modelling. Based on the calculated K D values, two populations of a2-adrenoceptor sites were distinguished: the high-affinity [3H]RX821002 binding sites (0.2 < KD (nM)< 0.6) and the low-affinity [3H]RX821002 binding sites (4.0 < KD (nM) <8.5). Values are the means_+S.E, from (n) experiments

[3H]RX821002 bound to a single population of non-interacting sites. Analysis of the data under the assumption of two sites was not statistically valid compared to a model assuming one site. The apparent dissociation constant values (K D) are summarized in Table 1. In membrane homogenates from tissues, Scatchard transformation of saturation data from liver extracts showed a single population of binding sites (Fig. 2B). Conversely, Scatchard analysis of saturation data from whole brain and kidney extracts indicated that a twosite model approximated the data better. Fitting the data to a two site-model resulted in a significant reduction in the sums of squares as compared to fitting the data to a one-site model (P < 0.05). Scatchard transformation of the saturation data (Fig. 2B) showed a curvilinear profile, indicating the binding of [3H]RX821002 to two distinct populations of sites. The K D values of

~

~ u

g

3.1.2. C o m p e t i t i o n s t u d i e s

To further characterize the c~2-adrenoceptor populations identified by [3H]RX821002 binding, yohimbine and prazosin ( a - a d r e n o c e p t o r antagonists) and oxymetazoline (a2-adrenoceptor agonist) were tested in competition studies with a fixed concentration of the radioligand. The data of the competition experiments were analysed by computer modelling. In isolated cells, computer analysis indicated that yohimbine, prazosin and oxymetazoline displaced [3H]RX821002 from a single population of sites. The K i values for yohimbine and prazosin are indicated in Table 1. The K i values for oxymetazoline calculated from three separate experiments were 3 + 1 nM and 41 _+ 13 nM in adipocytes and in colocytes, respectively. In whole tissue extracts, competition curves obtained with prazosin and yohimbine were monophasic in liver homogenates. They were clearly biphasic in kidney and brain homogenates (Fig. 3), whereas the competition curves for oxymetazoline were slightly shallow (not shown). In kidney and brain, computer modelling indicated the presence of two heterogenous populations when a two-site model and one-site model (P < 0.05) were compared for the competition with yohimbine and prazosin. The K i values are shown in Table 1. There was a 30-fold difference in the yohimbine affinities between the two c~2-adrenoceptor sites in the kidney and a 57-fold difference in the brain. For prazosin, the difference was 23-fold in the kidney and 38-fold in the brain. With oxymetazoline, the one-site inhibition model fitted better in liver, kidney and brain. The K i values calculated from three separate experi-

lOOI

KIDN13Y

8°]

100

[3H]RX821002 for both sites were calculated and are indicated in Table 1. Computer analysis showed that there was a 22-fold difference in the [3H]RX821002 affinities towards the two populations identified in the kidney and a 27-fold difference in the brain.

80-

60

60.

40

40-

2

20.

-11-10-9

-8

-7

-6

-5

.-4-3

-2

o -11-10-9

-8

-7

-6

-S

-4-3

-2

Concenu'ation of c o m p e t i t o r log(M)

Fig. 3. Inhibition of [3HlRX821002 binding to a2-adrenoceptor sites expressed in kidney and brain by yohimbine (o) and prazosin (e). Membranes prepared from kidney and brain were incubated in the presence of 6 nM [3H]RX821002 and the indicated concentration of competitor. Non-specific binding was defined in the presence of 10 - 4 epinephrine and the results are expressed as percent of control which corresponds to specific [3H]RX821002binding in the absence of competitor. Mean Ki values obtained from computer analysis of the curves are given in Table 1.

100

A. Bouloumi~et al. /European Journal of Pharmacology Molecular PharmacologySection 269 (1994) 95-103 -

m e n t s w e r e 123_+39 n M in liver, 290_+34 n M in k i d n e y a n d 105 _+ 85 n M in brain. A c c o r d i n g to t h e p r e v i o u s d e f i n i t i o n o f a z - a d r e n o c e p t o r s u b t y p e s , t h e high-affinity p o p u l a t i o n for [3H]RX821002, which is also t h e low-affinity site for y o h i m b i n e a n d p r a z o s i n , c o r r e s p o n d s to t h e r o d e n t a z A - a d r e n o c e p t o r subtype. T h e K D v a l u e for [3H] RX821002 as well as the y o h i m b i n e a n d p r a z o s i n K i v a l u e s of a z A - a d r e n o c e p t o r s in a d i p o c y t e s a n d colocytes w e r e well c o r r e l a t e d with t h e K D a n d the K i of t h e high affinity [3H]RX821002 b i n d i n g sites i d e n t i f i e d in k i d n e y a n d brain. In c o n t r a s t , the K D a n d t h e Ki v a l u e s f o u n d for t h e low affinity [3H]RX821002 sites i d e n t i f i e d in liver, k i d n e y a n d b r a i n d i d not c o r r e l a t e with t h o s e p r e v i o u s l y d e f i n e d for any of t h e o t h e r a z A - a d r e n o c e p t o r sites.

3.2. Description of rodent a2A-adrenoceptor m R N A distribution T h e specificity o f t h e u n i f o r m l y l a b e l l e d a n t i s e n s e c R N A p r o b e was t e s t e d . N o signal was d e t e c t e d with R G 2 0 o r a2-C10 D N A f r a g m e n t s in R N a s e - p r o t e c t i o n assays (not shown), w h e r e a s single s p e c i e s o f t r a n scripts w e r e p r o t e c t e d f r o m R N a s e in h a m s t e r tissues a n d cells (Fig. 4). T h e s e results d e m o n s t r a t e t h e strict specificity o f t h e s y n t h e s i z e d p r o b e for t h e h a m s t e r a 2 A - a d r e n o c e p t o r transcripts. In i s o l a t e d cells, a d i p o c y t e s a n d colocytes, specific h y b r i d i z a t i o n s w e r e d e t e c t e d (Fig. 4A) like in w h o l e k i d n e y a n d b r a i n (Fig. 4B). N o signal was i d e n t i f i e d in w h o l e liver. T h e s e results show t h a t a z A - a d r e n o c e p t o r

A: CELLS Adip +

C

T

Colo C T

B: TISSUES Kidney Brain Liver C T

C T

Table 2 a2-adrenoceptor densities (Bmax), expressed in fmol/mg protein, determined by computer modelling of [3H]RX821002 binding to kidney, brain and liver membranes Bmax Control

Bma x + Testosterone

High-affinity [ 3H]RX821002 binding sites Kidney 5 _+ 2 (4) 9_+ 2 (3) Brain 74+ 9 (3) 96-+ 13 (3) Low-affinity [3H]RX821002 binding sites Liver 24-+ 3 (5) 18-+ 2 (4) Kidney 30-+ 5 (4) 40_+11 (3) Brain 147 -+26 (3) 127-+ 9 (3) Whole extract membranes were prepared from control or testosterone-treated male hamsters (1 mg/day/animal for one week). Based on the calculated K o values, two populations of ae-adrenoceptor sites were distinguished: the high-affinity [3H]RX821002 binding sites (0.2 < K D (nM) < 0.6) and the low-affinity [3H]RX821002 binding sites (4.0 < K D (nM) < 8.5). Student's t-test was performed to compare the means_+S.E, of (n) separate experiments between control and treated animals.

t r a n s c r i p t s a r e e x p r e s s e d in a d i p o c y t e s , colocytes, kidney a n d b r a i n a n d a r e c o n s i s t e n t with the b i n d i n g data.

3.3. Regulation of a2-adrenoceptor expression in various cells and tissues U n d e r c h r o n i c t e s t o s t e r o n e t r e a t m e n t of m a l e h a m sters ( a d m i n i s t r a t i o n o f 1 m g of t e s t o s t e r o n e / d a y / a n i m a l for 7 days), analysis of [3H]RX821002 b i n d i n g s h o w e d n o m o d i f i c a t i o n o f t h e a p p a r e n t dissociation c o n s t a n t s in t h e various tissues ( d a t a not shown). Testosterone treatment did not alter the a2-adrenoc e p t o r d e n s i t i e s (Bm~x) d e t e r m i n e d a f t e r c o m p u t e r analysis o f t h e s a t u r a t i o n studies in w h o l e m e m b r a n e extracts f r o m kidney, b r a i n o r liver ( T a b l e 2). In isol a t e d cells, no m o d i f i c a t i o n of the a z A - a d r e n o e e p t o r

C T 1000 x

-=

m~ ¢~ o.

600

e~E

400

~'-.~ c~

200

o o

xz

Fig. 4. a2A-adrenoceptor RNA steady-state levels in adult male hamsters. (A) The unifornaly labelled hamster probe was hybridized, or not, with hamster amplified DNA fragment (lanes +, - ) , 50/zg of total RNA prepared from adipocytes (lanes Adip), 50 /~g of total RNA prepared from colocytes (lanes colo) from control or testosterone-treated hamsters (lanes C, T). (B) The uniformly labelled hamster probe was hybridized with 100 /zg of total RNA extracted from brain (lanes Brain), 200/xg of total RNA extracted from kidney (lanes Kidney) and 200 /zg of total RNA extracted from liver (lanes Liver) from control or testosterone-treated hamsters (lanes C and T). Representative autoradiograms of the electrophoresis of the samples digested with a mixture of RNase A and T1 are shown.

800

0 Adipocyte

Colocyte

Fig. 5. a2-adrenoceptor densities (Bmax), expressed in fmol/mg protein, determined by computer modelling of [3H]RX821002 binding in adipocyte and colocyte membranes isolated from control (open) or testosterone-treated (black) male hamsters (1 mg/day/ animal for one week). Student's t-test was performed to compare means -+S.E. of four separate experiments between control and treated animals; **P < 0.0t.

A. Bouloumid et al. /European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 95-103 !

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number was observed in colocytes, whereas in adipose ceils a significant increase (P < 0.01) in the a2A-adrenoceptor density was noted (Fig. 5). Thus, the androgenic regulation of a2-adrenoceptor expression was specific to the a2A-adrenoceptor subtype expressed in adipose tissue, since no modification was observed either in tissues expressing another subtype (kidney, brain and liver) or in cells and tissues expressing the same subtype (kidney, brain, colocytes). It was noticeable that an increase in a2A-adrenoceptor mRNA levels, identified 'by RNase protection assay, was only seen in adipocytes following testosterone administration (Fig. 4).

cellular distribution of the androgen receptors showed a greater binding to nucleus than to cytosol (18 + 5 fmol/mg protein on nuclear extracts and 4.0 + 1.6 fmol/mg protein on cytosolic extracts, n = 3, P < 0.05). Routinely, studies on cells and tissues were carried out on a total extract and androgen receptors were quantified using a saturable concentration of radioligand (10 nM) (Fig. 6B). Brain, kidney, and colocytes exhibited specific [3H]R1881 binding, whereas no binding was detectable in liver.

3.4. Expression of the androgen receptor in various cells and tissues

Alpha2-adrenoceptors provide a model of a heterogeneous population of proteins expressed in multiple tissues, encoded by different genes and which exhibit distinct pharmacological properties and trigger various signal transduction mechanisms when activated by catecholamines. The involvement of androgens had been previously described in the regulation of a2-adrenoceptor expression in adipose tissue during the sexual maturation of male hamsters (Saulnier-Blache et al., 1992). Considering the large heterogeneity of o~2adrenoceptor distribution, function and subtype, we hypothesized that there may be subtype-specific responses to androgens in various tissues, for instance in adipose tissue, kidney, brain, liver, and colon. Physiological studies first demonstrated the diversity of the functions mediated by activation of a2-adrenoceptors in both the central nervous system and the peripheral tissues (for review, Ruffolo et al., 1991). Pharmacological and molecular studies confirmed this heterogeneity by showing the existence of at least three different subtypes of receptor proteins, called a2A, a2B and a2c corresponding to three genes cloned in man: a2-C10, C2 and C4, respectively (Bylund et al., 1992). Recently, interspecies variation in yohimbine binding properties (az-adrenoceptor antagonist) has been described for the a2A subtype (Link et al., 1992). The corresponding gene (RG20) was cloned in rat (Lanier et al., 1991) and mouse (Link et al., 1992). The first 100 or so amino acids, deduced from the hamster a2-adrenoceptor fragment cloned in the present study by Polymerase Chain Reaction, exhibited a high degree of homology with the RG20 and ot2-C10 sequences (> 96%) whereas low homology was observed when comparing with a2B and azc sequences (< 84%, < 72%, respectively). Thus, the fragment of the cloned hamster a2-adrenoceptor corresponded to a species-specific variation of the a 2C10 adrenoceptor, as for the rat RG20. Pharmacological studies of az-adrenoceptor distribution with the radioligand [3H]RX821002 in hamster tissues show that [3H]RX821002 exhibits different affinities towards the az-adrenoceptor subtypes, allowing the definition of the tissue distribution of the

Androgen receptors were quantified to determine the androgen receptivity in various tissues and cells. Studies of non-linear regression and Scatchard analysis were carried out on adipocyte nucleus extracts. [3H] R1881 exhibited saturable and specific binding. The binding parameters are indicated in Fig. 6A. The sub-

A

0 0

30

10 20 30 40 [3H]R1881 Specific Bound (fmol/mg protein)

B

v T

-Io m

lO

0 Adlpocyte Colocyte

KIdne

Brain

Liver

Fig. 6. (A) Typical Scatchard transformation of [3H]R1881 binding data on adipocyte nucleus extracts from one representative experiment. The m e a n dissociation constant from three separate experiments ( K D) was 2.5+0.8 nM. (B) Specific [3H]R1881 binding ( f m o l / m g protein) determined in total extracts of adipocytes, colocytes, kidney, brain and liver of adult male hamsters. Values are m e a n s +_S.E. of at least four separate experiments.

4. Discussion

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A. Bouloumi~ et al. / European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 95-103

a2-adrenoceptor subtypes. Some tissues exhibited a single az-adrenoceptor population (adipocytes, colocytes and liver), whereas others exhibited at least two (kidney and brain). The binding properties of the a2adrenoceptors expressed in adipocytes and colocytes for various drugs were identical and corresponded to the rodent O/2A subtype which exhibited a high affinity for the radioligand [3H]RX821002 and oxymetazoline and a low affinity for prazosin and yohimbine. These properties were common to a single population of a2-adrenoceptor sites identified in kidney and brain; a result showing that the a2A subtype was also present in hamster kidney and brain, as previously demonstrated in the rat (Uhlen and Wilkberg, 1991; Uhlen et al., 1992). These results were confirmed by a2A-adrenoceptot mRNA distribution studies, which displayed specific hybridization of the hamster a2A-adrenoceptor probe in adipocytes, colocytes, kidney and brain. Previous studies in the rat have described the coexistence of Og2Aand c~2B subtypes in the kidney (Uhlen and Wilberg, 1991). The pharmacological definition of an O/2Bsubtype is consistent with our data (high affinity for yohimbine and prazosin, and low affinity for [3H]RX821002), suggesting that a2A- and azB-adrenoceptor subtypes exist together in hamster kidney. The a2-adrenoceptor identified in the liver exhibits pharmacological properties similar to those of a2~ subtype. Since specific hybridization with o12-C2 probe has been demonstrated in rat liver (Lorenz et al., 1990), it could be suggested that the a2-adrenoceptor expressed in hamster liver may also be an aZB related adrenoceptor subtype. Previous studies on the rat central nervous system had shown that the brain displayed a differential distribution of all three a2-adrenoceptor subtype mRNAs according to the anatomical location (Nicholas et al., 1993). In the present study, only two distinct populations were identified by binding studies; one was identified as being the a2A subtype. The precise delineation of the other population appeared to be difficult since it may reflect the coexistence of the both a2~ and a2c subtypes. Further delineation of the non-A c~2adrenoceptors was considered to be out of the scope of the present paper. Chronic testosterone administration did not modify the binding of the radioligand to the a2-adrenoceptors expressed in the tissues studied. No changes in a2A- or non-A aE-adrenoceptor densities were observed in colocytes, kidney, liver or brain. Only in adipocytes was a2A-adrenoceptor number increased after testosterone treatment. These results show that testosterone specifically up-regulates the expression of the adipocyte a2Aadrenoceptor. The generally described pathway of signal transduction mediated by steroids involves specific binding to cytoplasmic or nuclear receptors (for review, King, 1992). When formed, the complex is able to bind to specific DNA sequences (steroid response elements)

located upstream in the promoting region of the genes controlling the transcription. Previous studies had shown, with Northern blotting, a testosterone effect on the amount of adipocyte a2-adrenoceptor mRNAs (Saulnier-Blache et al., 1992), which was also observed in the present study with RNase protection assay specifically on adipocyte a2A-adrenoceptor mRNA levels. Whether the adipocyte az-adrenoceptor response is mediated through an interaction of the ligandactivated androgen receptor via an androgen response element remains to be established. However, an explanation for the lack of effect of testosterone on the non-adipocyte az-adrenoceptor through a lack of androgen receptivity can be ruled out inasmuch as [3H]R1881 receptors were identified in all the tissues studied, except the liver. The precise molecular mechanisms involved in this androgen-specific regulation of adipocyte a2A-adrenoceptor have now to be defined in an appropriate cell model in order to further characterize the components involved in the adipose tissue specificity. References Bonne, C. and J.P. Raynaud, 1975, Assay of androgen binding sites by exchange with methyltrienolone (R1881), Steroids 27. 497. Bylund, D.B., H.S. Blaxall, L.J. Iversen, M.G. Caron, R.J. Lefkowitz and J.W. Lomasney, 1992, Pharmacological characteristics of o~2-adrenergic receptors: comparison of pharmacologically defined subtypes with subtypes identified by molecular cloning, Mol. Pharmacol. 42, 1. Bylund, D.B., 1992, Subtypes of at- and a2-adrenergic receptors. FASEB J. 6, 832. Chalberg, S.C., T. Duda, J.A. Rhine and R.K. Sharma, 1990, Molecular cloning, sequencing and expression of an alpha-2-adrenergic receptor complementary DNA from rat brain, Mol. Cell. Biochem. 97, 161. Chomczynski, P. and N. Sacchi, 1987, Single step method of RNA isolation by acid guanidinium thiocyanate-Phenol-chloroform extraction, Ann. Biochem. 162, 156. Eason, M.G. and S.B. Liggett, 1992, Subtype-selective desensitization of a2-adrenergic receptors, J. Biol. Chem. 267, 25473. Guyer, C.A., D.A. Horstman, A.L. Wilson, J.D. Clark, E.J. Cragoe and L.E. Limbird, 1990, Cloning, sequencing, and expression of the gene encoding the porcine alpha-2-adrenergic receptor: allosteric modulation by Na +, H +, and amiloride analogs, J. Biol. Chem. 265, 17307. King, R.J.B., 1992, Effects of steroid hormones and related compounds on gene transcription, Clin. Endocrinol. 36, 1. Kobilka, B.K., H. Matsui, T.S. Kobilka, T.L. Yang-Feng, U. Francke, M.G. Caron, R.J. Lefkowitz and J.W. Regan, 1987, Cloning, sequencing, and expression of the gene coding for the human platelet a2-adrenergic receptor, Science 238, 650. Laburthe, M., B, Amiranoff and C. Boissard, 1982, Alpha-adrenergic inhibition of cyclic AMP accumulation in epithelial cells isolated from rat small intestine, Biochem. Biopbys. Acta 721, 1111. Lanier, S.M., S. Downing, E. Duzic and C.J. Homey, 1991, Isolation of rat genomic clones encoding subtypes of the a2-adrenergic receptor, J. Biol. Chem. 266, 10470. Link, R., D. Daunt, G. Barsh, A. Chruscinski and B. Kobilka, 1992, Cloning of two mouse genes encoding a2-adrenergic receptor subtypes and identification of a single amino acid in the mouse

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