Differential modulation of α2-adrenoceptor subtypes in rat kidney by chronic desipramine treatment

PII s0024-3205(99)00186-1

ELSEVIER

Life Sciences, Vol. 64, No. 2.5, pp. 2327-2339, 1999 copyright 0 1999 Ebmier science Inc. Printed in the USA. All rights reserved 0024-3205/59/Ssee front matter

DIFFERENTIAL MODULATION OF a2-ADRENOCEPTOR SUBTYPES IN RAT KIDNEY BY CHRONIC DESIPRAMINE TREATMENT Luis F. Callado, Ane M. Gabilondo and J. Javier Meana Department

of Pharmacology,

University

of the Basque Country, E-48940 Leioa, Bizkaia, Spain

(Received in final form March 1, 1999)

Summary The profile of [3H]RX821002 (2-methoxy idazoxan) binding to a+drenoceptor subtypes in rat kidney membranes was evaluated in controls and after chronic treatment with desipramine (10 mg/kg, i.p., every 12 h, 7 days) or clorgyline (2 mg/kg, i.p., every 24 h, 21 days). [3H]RX821002 recognized with high afftnity &=1.5*0.2 nM in controls) a single and saturable population of binding sites (B,,=57&5 finol/mg protein in controls). The competitions by (-)-adrenaline, the azn-adrenoceptor selective drug ARC239 (2-[2-[4-(o-methoxyphenyl)-piperazin-lyl]-ethyl]-4,4-dimethyl-l,3(2H,4H)-isoquinolindione) and the a2A-adrenoceptor selective drug BRL44408 (2-[2H-( 1-methyl-1,3-dihydroisoindole)methyl]-4,5dihydroimidazole) suggested the existence of both a2A- and aza-adrenoceptors together with a non-adrenoceptor binding site. After chronic desipramine but not after chronic clorgyline treatments, the density (B,,,& of az-adrenoceptors was increased (46%). In the presence of ARC239 (50 nM), the density of a2A-adrenoceptors increased (44%) in the desipramine-treated group without changes in the clorgyline-treated group. Conversely, in the presence of BRL44408 (100 r&L), the density of a2aadrenoceptors was not affected by the treatments. The selective upregulation of the oz.&-adrenoceptor subtype following chronic desipramine administration is compatible with a differential location and function of the a@lrenoceptor subtypes in the rat kidney.

Key Words: a2-adrenoceptor subtypes, desipramine, clorgyline, kidney

Evidence of multiple az-adrenoceptor subtypes has been provided on the basis of pharmacological and molecular criteria (1). Using molecular cloning, radioligand binding studies and fi.WtiOIIal assays, three subtypes of a2-adrenoceptors, called UZA,ala, and azc-adrenoceptors, have been identified in rats (2,3). The in vivo functional role of the different a2-adrenoceptor subtypes has been recently evaluated by gene disruptions or mutations of the all three members of the a&renoceptor subfamily (4,5).

Corresponding author (present address): Dr. Luis F. Callado. Neurotmnsmission Anaesthetics Unit. The Royal London Hospital. London El IBB, United Kingdom. Fax +44-171-377-7126. Email: [email protected]

Lab.

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Multiple az-adrenoceptor subtypes are expressed in the rat kidney (6-10) where the a2a-adrenoceptor is considered as the predominant subtype (7,8,10-12). Recently, it has been suggested that this subtype could be involved in the control of free water clearance (13,14). The az*-adrenoceptor subtype is also present in rat kidney where it seems to influence the osmolar clearance (solute and sodium excretion) by indirect mechanisms (13). Many functional lines of evidence indicate that the az*-adrenoceptor is also the subtype that modulates the release of noradrenaline from sympathetic nerve terminals in the rat kidney ( 15 17). Desensitization of central and peripheral al-adrenoceptors in response to chronic treatment with various antidepressant drugs has repeatedly been reported (18-22). The modulation of az-adrenoceptors by these antidepressant drugs involves the elevation of synaptic noradrenaline concentrations leading to enhanced receptor activation after the acute treatment followed by receptor densensitization with long-term administration (20). Since, in rat, the az*-adrenoceptor is more widely expressed than the a;?a-adrenoceptor (5,6,23), the above proposed aI-adrenoceptor downregulation has been mainly demonstrated for the a2A-adrenoceptor subtype. In cell cultures and after exposure to the endogenous agonists noradrenaline or adrenaline, a subtype-selective desensitization has been described (24-26) suggesting different adaptative mechanisms of the a2-adrenoceptor subtypes that have not yet been explored. The present study was designed to evaluate, in the same tissue, the biochemical characteristics of the different az-adrenoceptor subtypes after in vivo long-term treatment with the selective noradrenaline uptake blocker desipramine or the monoamine oxidase inhibitor clorgyline. The methoxy analog of idazoxan [3H]RX821002 was chosen as a suitable tool to quantify the biochemical parameters of the a2A- and aza-adrenoceptor subtypes in rat kidney (7,8,10). This radioligand has been found to recognize both a2A- and ala-adrenoceptors in rat kidney (7,8,10,27) and other tissues (28-32).

Methods Animals and treatments. Male Sprague-Dawley rats (final weight 230-270 g) were maintained on a 12: 12 h light/dark cycle with free access to food and water. In experiments designed to examine the effects of antidepressant drugs, the animals were treated with either desipramine (10 mg/kg, i.p., every 12 h) for 7 days or clorgyline (2 mg/kg, i.p., every 24 h) for 21 days. During the same periods, control groups received saline (1 ml/kg, i.p.) treatment. 24 h after the last injection of drugs the rats were killed and their kidneys were excised and stored at -70°C until assays were performed. The doses of desipramine and clorgyline were considered optimal for the modulation of az-adrenoceptors on the basis of previous reports (20,33). Membrane preparation. Membranes were prepared as previously described (33). After thawing, the kidneys were homogenized in 5 ml of ice-cold Tris-sucrose buffer (5 mM Tris-HCl, 250 mM sucrose, 1 mM MgC12, pH 7.4) and centrifuged at 1,lOOxg for 10 min. The supematants were centrifuged at 40,OOOxg for 10 min and the resulting pellets washed and centrifuged twice with 2 ml of fresh Tris incubation buffer (50 mM Tris-HCl, 1 mM EDTA, 0.1 mM Gpp(NH)p (5’-guanylylimidodiphosphate), 140 mM NaCl, pH 7.5). The final pellets were resuspended in incubation buffer to a final protein concentration of 0.7-1.3 mg/ml. Protein content was determined by the method of Lowry et al. (34) with bovine serum albumin as the standard. Binding axsays. [3H]RX821002 binding assays were performed as described previously (10) by incubating, for 30 min at 25”C, 550 ~1 of a membrane preparation with [ HlRX821002 and

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different drugs. For the saturation studies, 0.13 nM to 16 nM of [3H]RX821002 (eight concentrations) were used under standard conditions whereas the competition studies were performed by incubation of the membranes with [3H]RX821002 (2 r&l) in the absence or presence of various drug concentrations (1 O-l2 M to 10m3M; 19 concentrations). Incubations were stopped by dilution of the samples with 5 ml of ice-cold Tris incubation buffer (4°C) and then filtered and washed on Whatman GF/C glass fiber filters (Brandel cell Harvester) presoaked with 0.5% polyethylenimine. The filters were transferred to minivials containing 5 ml of OptiPhase “HiSafe” II cocktail (Wallac, UK) and counted for radioactivity for 5 min by liquid scintillation spectrometry (Packard 2200CA). Analyses of binding data. Analyses of individual saturation isotherms (&, dissociation constant; B,,, maximum number of binding sites) and competition experiments (K;, inhibition constant) were performed by computer-assisted nonlinear regression using the EBDA-LIGAND programs (35,36). Saturation curves of control and antidepressant-treated groups were also simultaneously coanalyzed (all experiments of each group being analysed together). To determine the best fit, a one-site model of radioligand binding was initially assumed and then a two- or more site binding models were tested (37,38). The selection between different binding models was made statistically using the principle of extra sum of squares (F test) and the more complex model was accepted if the resulting P value was less than 0.05. The nonparametric Kruskal-Wallis and Mann-Whitney U-tests were used for statistical comparisons between groups, assuming a level of significance of PcO.05. A simultaneous analysis of experiments was also performed to determine whether the control and antidepressanttreated groups differed significantly from each other. First, the two sets of data (antidepressanttreated and control groups) were analyzed separately. The overall value for the sum of squares and the degrees of freedom was the sum of the individual values from each fit. Next, the two sets of data were pooled and analyzed simultaneously constraining them to share one or more common parameters (I(d, B,,). The pooled fit yielded values for the sum of squares and degrees of freedom. The analysis that permitted one or more of the parameters to be shared without a significant increase in the residual variance was taken as the best fit (37,38). The statistical significance of the improvement was determined by an F test with a level of significance of PCO.05. Results are expressed as individual meanhS.E.M. from n separate experiments performed in membranes from different animals or as the best fit value&tandard errors (SE.) as determined by the computer program. The S.E. values obtained from simultaneous nonlinear regression were not used in further formal statistical calculations. Isotopes, drugs and chemicals. [3H]RX82 1002 (1 ,4-[6,7(n)-[3H]benzodioxan-2-methoxy-2-yl)-2imidazoline HCl; specific activity 53-62 Ci/mmol) was purchased from Amersham International (Amersham, UK) and stored at 4°C. For the assays, appropriate amounts of the stock solutions were diluted with purified water (Milli-RO, Mill&Q) containing 2.5 mM HCl and 6% ethanol. (-)-Adrenaline bitartrate and desipramine HCl were from Sigma Chemical Co. (St. Louis, MO, USA); 2-[2 -[4-(o-methoxyphenyl)-piperazin-I-yl]-ethyl] -4, 4- dimethyl-1, 3(2H, 4H) isoquinolindione HCl (ARC239) was from K Thomae GmbH (Biberach, Germany), 2-[2H-(l-methyl-l,3-dihydroisoindole)methyl]-4,5-dihydroimid~ole (BRL44408) was from Beecham (Essex, UK); clorgyline HCl was from RBI (Natick, MA, USA). All other chemical reagents were of analytical quality and were purchased from Merck (Darmstadt, Germany) or Sigma Chemical Co. For the chronic treatments all the drugs were dissolved in a 0.9% NaCl (saline) solution.

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Results [:‘H]RX821002 bindin sites in rat kidney. In low&in&y conditions for agonists, the B competition curves of [ HlRX821002 binding (2 nM) by (-)-adrenaline showed a biphasic pattern (Kin=71*25 nM, KiLz7.37k1.36 PM; n=3) in naive (non-treated) rats. In the presence of ARC239 (50 nM) to selectively mask the a2s-adrenoceptor subtype (for further details, see reference 10) (-)-adrenaline still displayed biphasic competition curves (Kin=27l*lO5 nM, KiLT15.9f1.6 PM; n=2). Similarly, the competition curves of [3H]RX821002 binding by (-)-adrenaline in the presence of BRL44408 (100 nM) to selectively mask the a2A-adrenoceptor subtype, were also biphasic (Kin=191*4O nM, KiLz15.3k1.4 PM; n=2). All these previous data reflect the presence of a2A- and a2aadrenoceptors and a non-adrenoceptor binding site in rat kidney membranes, as previously reported (10). Consequently, in subsequent saturation experiments for the evaluation of a&renoceptors, (-)-adrenaline (1 PM) was used to delineate the non-adrenoceptor [3H]RX821 002 binding into the nonspecific binding component.

Parameters of [3H]RX821002 binding to az-adrenoceptors in control rats and rats chronically treated with desipramine and clorgyline. In order to study the effect of chronic antidepressant treatment on the biochemical characteristics of a2-adrenoceptors in rat kidney, saturation curves of (-)-adrenaline displaceable [3H]RX821002 binding were obtained. The specific binding of [3H]RX821002 in controls and antidepressant-treated rats comprised high-affinity labelling of a single saturable population of sites (Table I). The density (Bmaxvalues) of a2-adrenoceptors in the group of rats treated with desipramine was 46% higher than in control rats (Table I). In contrast, the density of a2-adrenoceptors after chronic clorgyline treatment was similar to that obtained in the control group (Table I).

TABLE I Parameters of Saturation Experiments with [3H]RX821002 to az-Adrenoceptors in Kidney Membranes from Control Rats and Rats Chronically Treated with Desipramine and Clorgyline. Kd

B *ax

n

W)

(fmol/mg protein)

Control

1.5hO.2

57*5

12

Desipramine

1.7ztO.2

83*6a

8

Clorgyline

1.6hO.2

56*4

8

Rat kidney membranes were incubated at 25°C for 30 min with eight concentrations of [3H]RX821002 (0.13-l 6 r&i). Specific binding was determined by subtracting nonspecific binding (defined with 1 @I (-)-adrenaline) from total binding. The dissociation constants (&) and the maximum number of binding sites (B,,.,& were determined by nonlinear analysis using the EBDA-LIGAND programs. Group values are mean&S.E.M. of n experiments. *P
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The simultaneous analysis of saturation curves from control and desipramine-treated groups (all experiments together) indicated the existence of differences between both groups (F[2,189]=18.5; P
(10 mg/kg, i.p., two doses, n=3) or clorgyline a 24 h period of drug washout did not induce any from acutely saline (1 ml/kg, one dose, n=3) treated 1.450.2 nM; B ,,,* values = 6ti4, 5sK3 and 64*5 and clorgyline treatments, respectively).

[3H]RX821002 (nM) Fig. 1 Specific binding of r3H]RX82 1002 to qadrenoceptors in kidney membranes from control rats (0) and rats treated with desipramine (10 mg/kg, i.p., every 12 h, for 7 days) (0) and clorgyline (2 mg/kg, i.p., every 24 h, for 21 days) (A) as a function of increasing concentrations of the radioligand. Each point is the mean*S.E.M. of n cases shown in Table I. Analysis of saturation isotherms from the various groups yielded in control rats (0) an hyperbolic plot with a & of 2.lkO.4 nM with a B, of 64*4 fmol/mg protein. In rats treated with desipramine (o), the hyperbolic plot yielded a ILJ of 3.8*0.7 r&I with a B ,,,= of 97*7 finol/mg protein. In rats treated with clorgyline (A), the hyperbolic plot yielded a Kd of 2.6hO.3 nM with a B,, of 66*3 finol/mg protein. Nonspecific binding was determined in the presence of (-)-adrenaline (1 PM).

Competition of fH]R&821002 binding to a2-adrenoceptors subtypes by selective drugs in control rats and rats chronically treated with desipramine and clorgyline. To confirm that the concentrations of ARC239 and BRL.44408 used to selectively mask theaz-adrenoceptor subtypes in drug-free animals were also suitable for a further masking in drug-treated groups, competition assays by (-)-adrenaline, ABC239 and BRL44408 were performed under the different conditions.

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curves of [3H]RX821002 total binding by the adrenergic drugs (-)-adrenaline, ARC239 and BRL44408 were biphasic and compatible with the presence of two az-adrenoceptor subtypes and a non-adrenoceptor binding site. (-)-Adrenaline was able to discriminate between the a2-adrenoceptor component (high affinity site) and the catecholamine-insensitive component (low affinity site) of [3H]RX821002 binding, while ARC239 displaced with high affinity the aza-adrenoceptor subtype component and BRL44408 displaced with high affinity the a2*-adrenoceptor subtype component (Table II, Fig. 2). Nonspecific binding (N) values estimated by the curve-fitting program did not differ from those obtained experimentally in presence of 10 uM unlabelled RX82 1002 (10) and were similar among the different drugs (-3%). The competition

After chronic desipramine treatment, the potencies (Ki values) of (-)-adrenaline, ARC239 and BRL44408 did not differ from those in controls (Table II, Fig. 2). An increase in the high affinity proportion for BRL44408 was observed after chronic desipramine treatment. In the group of rats treated with clorgyline, the competition curves by (-)-adrenaline, ARC239 and BRL44408 were aligned to control curves in both afftnity sites (Table II, Fig. 2). Thus, these antidepressant treatments did not modify the affinity of (-)-adrenaline, BRL44408 for the different [3H]RX821002 binding sites in rat kidney.

ARC239 and

TABLE II Parameters of Competition Experiments with [3H]RX821002 Binding by Adrenergic Drugs in Control Rats and Rats Chronically Treated with Desipramine and Clorgyline. (-)-Adrenaline

ARC239

H

n

KIH

(a) Control Desipramine Clorgyline

KiL

BRL44408 H

W)

W)

n

K,u W)

83*9

8.4kO.8

56*2

5

3.1h2.2

l.l*O.l

33*1

5

258*57

8.WO.5

5W4

3

4.6k2.5

1.1+0.2

29+6 4

83xt17

6.W0.3

57&l

5

O.S*O.l

1.2hO.2 26*5

4

KiL (PM)

H W)

n

47*28

0.7*0.1

33+12 3

8%6

1.8*0.3

69*3a 3

55*27

0.7*0.1

33*8

Rat kidney membranes were incubated at 2S’C for 30 min with [3H]RX821002 (2 nM) in the absence or presence of the different drugs (10-‘2-10-3 M; 19 concentrations). Experimental data were fitted by nonlinear regression using the EBDA-LIGAND programs. For each experiment, competition data were analyzed assuming initially a one-site model of [3H]RX821002 binding and then assuming two- or more site binding models. The selection between models was made statistically using the extra sum of squares principle (F test). The competition curves fitted the best to a two-site binding model. & values of [3H]RX821002 binding to both sites were fixed to 1 nM. Nonspecific binding (N) was allowed to float and to be adjusted by the curve-fitting program in order to obtain the best estimate for each set of experimental data. Note that under such conditions, [3H]RX82 1002 specific binding to az-adrenoceptors and to non-adrenoceptor binding sites was considered. Kin and KiL represent the inhibition constants against the high- (H) and low- (L) affinity sites. The proportion of highaffinity binding sites is represented by H (%). Values are meanstS.E.M. of n experiments. ‘P
3

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-10

-12 -If

-9

-8

-7

(-)-ADRENALINE

-6

-5

-4

-3

-4

-3

log [M]

100

80 60 40 20 0 -12

-11

-10

-9

-8

-7

-6

-5

ARC239 log [M’j

-12 -11 -10

-9

-8

-7

-6

-5

-4

-3

BRL44408 log [Mj Fig. 2

Competition curves of (-)-adrenaline (A), ARC239 (B) and BRL44408 (C) for [3H]RXS21002 (2nM) binding sites in kidney membranes from control rats (0) and rats treated with desipratnine (10 mg/kg, i.p., every 12 h, for 7 days) (0) and clorgyline (2 m@g, i.p., every 24 h, for 21 days) (A). Each point is the me&S.E.M. of n cases shown in Table II. The lines correspond to the computerassisted curve-fitting of all competition data from each experimental group (3-5 experiments analysed simultaneously) to the appropriate binding model. Analysis of the separate experiments by non-linear curve-fitting gave the binding parameters shown in Table II. For additional details see Table II.

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Parameters of [3HJRX821002 binding to az-adrenoceptor subtypes in control rats and rats chronically treated with desipramine and clorgyline. Biochemical characteristics of az*-adrenoceptors were quantified by saturation curves of (-)-adrenaline displaceable pHlRX821002 binding in the presence of ARC239 (50 nM) to selectively mask the aza-adrenoceptor subtype. After the inclusion in the assay of 50 nM ARC239, chronic treatment with desipramine markedly increased (44%) the density of a2a-adrenoceptors (Table III). In contrast, chronic treatment with clorgyline did not modify the density of a2A-adrenoceptors (Table III). Conversely, in the presence of 100 nM BRL44408, the densities of aza-adrenoceptors were not affected by chronic treatments with desipramine or clorgyline (Table III). The aza-adrenoceptor subtype showed lower affinity values than the az*-adrenoceptor subtype and these were not affected by the chronic antidepressant treatments (Table III). The simultaneous fitting of saturation data in the presence of ARC239 (50 nM) confiied the presence of differences in a2A-adrenoceptor parameters between control and desipramine-treated groups (F[2,40]=88.0; P
TABLE III Parameters of Saturation Experiments with [3H]RX82 1002 to az-Adrenoceptor Subtypes in Kidney Membranes from Control Rats and Rats Chronically Treated with Desipramine and Clorgyline. a2n-Adrenoceptors

a2A-Adrenoceptors Kd

B “W

n

Kd

B max

n

(r&I)

@nol/mg protein)

3

3.4*0.4

41*1

3

56*2a

3

2.5*0.1

421

3

41k3

3

5.6kO.l

4W2

3

(nM)

(fmol/mg protein)

Control

1.3rtO.l

3W2

Desipramine

1.4hO.l

Clorgyline

1.4kO.l

Rat kidney membranes were incubated at 25°C for 30 min with eight concentrations of [3H]RX821002 (0.13-l 6 r&I). Incubations were performed in the presence of ARC239 (50 nM) or BRL44408 (100 r&I) to selectively mask the a2a- and the a2Aadrenoceptor subtypes, respectively. Specific binding was determined by subtracting nonspecific binding (defined with 1 uM (-)-adrenaline) from total binding. The dissociation constant (IQ and the maximum number of binding sites (B-) were determined by nonlinear analysis using the EBDA-LIGAND programs. Group values are mean&S.E.M. of n experiments. ‘PcO.05 (Mann-Whitney U-test versus corresponding control).

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OY 0

2

4

6

8

10

12

[3H]RX821002 (nM)

4o1

0

0

B

2

4

6

8

10

[3H]RX821002 (nM) Fig. 3 Specific binding of [3H]RX821002 to azA-adrenoceptors (A) and ala-adrenoceptors (B) in kidney membranes from control rats (0) and rats treated with desipramine (10 mgkg, Lp., every 12 h, for 7 days) (13) or clorgyline (2 m&g, i.p., every 24 h for 21 days) (A) as a fknction of increasing concentrations of the radioligand. Experiments were performed in the presence of ARC239 (50 nM) for the evaluation of a2A-adrenoceptors (A) and in the presence of BRL44408 (100 nM) for the evaluation of aza-adrenoceptors (B). Each point is the meankS.E.M. of n cases shown in Table III. Analysis of saturation isotherms from the various groups yielded in control rats (0) an hyperbolic plot with a & of 2.3hO.3 nM with a B maxof47*3 finollmg protein for aza-adrenoceptors (A) and a Kd of 4.WO.7 nM with a B mByof 45i5 finol/mg protein for aza-adrenoceptors (B). In rats treated with desipramine (o), the hyperbolic plot yielded a Kd of 1.8*0.2 nM with a B,, of 62k2 fmol/mg protein for azl\-adrenoceptors (A) and a & of 3.6kO.S nM with a B,, of 44&4 finol/mg protein for aza-adrenoceptors (B). In rats treated with clorgyline (A),the hyperbolic plot yielded a K,j of 1.5kO.3 nM with a B,, of 423 fmol/mg protein for a2A-adrenoceptors (A) and a & of 5.7kO.8 nM with a B ,,,= of 46*4 fmol/mg protein for ala-adrenoceptors (B). Nonspecific binding was determined in the presence of (-)-adrenaline (1 PM).

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Discussion The present study evidences that striking differences exist among a&renoceptor subtypes in rat kidney with respect to their regulation by the antidepressant drugs desipramine and clorgyline. [3H]RX821002 represents an ideal ligand to label the total population of a~-adrenoceptors in rat kidney, due to its high affinity for the rat QA- and a2aadrenoceptor subtypes (7,8,3 1,39-41). The affinity of [3H]RX821002 at aXadrenoceptors has been proposed to be 2 to 7-fold higher than at aza-adrenoceptors (7,39,41,42). The presence of a single and saturable population of binding sites (Table I) confirms that the affinity values of [3H]RX821002 for a2A- and aan-adrenoceptors are very close in the rat kidney. In addition, the dissociation constants in the present report (Table III) are consistent with previous data and suggest that, under the present conditions, both subtypes can be labelled by the radioligand (7,8,10,43). The higher affinity of [3H]RX821002 for cQ,@drenoceptors tOgether with the preferential aan-adrenoceptor SekCtiVity (3 to lo-fold) of radioligands such as [3H]yohimbine and [3H]rauwolscine (8,11,12) could also explain the apparent conflict of the present results with studies suggesting a prevailing or exclusive presence of the aza-adrenoceptor subtype in rat kidney. In competition studies, both the azalc-adrenoceptor selective antagonist ARC239 and the a2A-adrenoceptor selective antagonist BRL44408 displayed biphasic curves (Table II), confirming that al,&- and ala-adrenoceptor subtypes are labelled by [3H]RX821002 in rat kidney (7,8,10). The ability of (-)-adrenaline to delineate in low-affinity conditions an heterogeneous binding of [3H]RX82 1002 to rat kidney membranes (Table II) is consistent with the hypothesis of a non-adrenoceptor site identified by the radioligand (10,44). Moreover, the selective masking of a2a-adrenoceptors by ARC239 (50 nM) or a&&-adrenoceptors by BRL44408 (100 nM) did not abolish the biphasic pattern of competition curves by (-)-adrenaline. Thus, the finding provides further evidence that a non-adrenoceptor [3H]RX821002 binding site is expressed in the rat kidney (10). Consequently, in the present study an optimal (-)-adrenaline concentration (1 PM) was chosen to delineate all the non-adrenoceptor component of the [3H]RX821002 binding into the nonspecific binding in saturation experiments (Tables I and III; Fig. 1 and 2). Therefore, [3H]RX821002 binding that could be displaced by (-)-adrenaline (1 PM) was assumed to be a2-adrenoceptors. The differential masking of aa-adrenoceptors with subtype-selective drugs represents a useful approach to estimate binding parameters when two or more subtypes are present in a tissue. However, the residual presence of antidepressant drugs or the treatments themselves could affect the affinity of the selective drugs for the different az-adrenoceptors and may modify the masking concentrations of the drugs needed in experimental groups under antidepressant treatment. Thus, it is important to evaluate the affinities under all treatment conditions and choose drug concentrations which have little masking effect on one subtype but block the maximal proportion of the other subtype. The present results indicate that the affinities of ARC239 and BRL44408 are not altered after chronic treatment with desipramine or clorgyline when followed by a washout period of 24 h (Table II). As consequence, 50 r&I of ARC239 and 100 nM of BRL44408 were selected as a suitable concentrations for the selective examinations of the a2-adrenoceptor subtypes (Table II). However, the antagonist BRL44408 displayed only a 13 to 22-fold selectivity among a2A- and aza-adrenoceptors (Table II) (7, lo), which does not allow the absolute masking of the a2A-adrenoceptor population while leaving unmasked the a2a-adrenoceptor population. This fact also explains the higher densities of al-adrenoceptors (CQA+ aTa) in Table III as compared with those in Table I. Despite this relative masking of azA&renoceptors by BRL44408, the modulatory effect induced by desipramine on the azA_adrenoceptor subtype could not be demonstrated in the remaining population. It is likely that the low abundance of unmasked a2Aadrenoceptors relative to the total az-adrenoceptor

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population under such a condition (mainly aza-adrenoceptors) changes in a2A-adrenoceptor density.

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does not allow one to detect the

az-Adrenoceptors as sites of catecholamine action undergo agonist-induced regulation of receptor density or affinity. A differential downregulation by agonist treatment has been reported between al-adrenoceptor subtypes expressed in cultured cells (24-26,45). Comparatively little is known about the in vivo regulation of each a&renoceptor subtype. In the rat kidney, a2A- and aza-adrenoceptor were clearly identified by [3H]RX821002 (Table III) allowing the study of selective regulation of both subtypes after systemic application of drugs known to affect renal noradrenergic activity (46-48). In the rat, chronic treatment with various antidepressant drugs results in a clear downregulation of central az-adrenoceptors (33,49-52) where the az,+-adrenoceptor is the predominant or exclusive subtype. This biochemical phenomenon has been strongly supported by functional studies where a central cx2-adrenoceptor desensitization after chronic antidepressant treatment has been observed (19,21,22,53,54). However, the findings are less conclusive in the peripheral nervous system (20,46,55). It could be argued that an enhanced a&&renoceptor density might reflect the residual presence of antidepressant drugs in the membrane preparation. However, the finding of unchanged & values of the [3H]RXS21002 binding (Tables I and III) makes such a competitive mechanism improbable. Moreover, 24 h is a well-established washout period to evaluate the modulation of az-adrenoceptors by chronic antidepressant treatments in central and peripheral nervous systems (20,52). The increased density of renal al-adrenoceptors obtained after desipramine treatment (Table I) should be considered as representative of a selective increase of a2A- but not aza-adrenoceptors (Table III). In contrast to desipramine, chronic clorgyline treatment did not induce density changes neither in a2A- nor in aza-adrenoceptors (Tables I and III). The lack of effect after chronic clorgyline treatment on a2-adrenoceptors cannot be ascribed to the use of low doses of the drug because clorgyline (1 mg.kg daily for 21 days) have demonstrated to induce downregulation of az-adrenoceptors in rat brain (52). Moreover, it is of interest to note that chronic clorgyline (1 mg/kg daily for 2 1 days) is able to down-regulate vascular ai-adrenoceptors with no change in vascular and cardiac a;?-adrenoceptors (46). Differences in the location and functions of the kidney az-adrenoceptor subtypes and their accessibility to circulating catecholamines may account for the observed results. Physiological studies suggest that rat renal sympathetic nerves possess presynaptic az-adrenoceptors that modulate the release of noradrenaline and belong to the a2A-subtype (15-l 7). In contrast, aza-adrenoceptors of kidney are mostly concentrated in the basolateral membranes of isolated tubules (56,57). In collecting tubules, a2-adrenoceptor activation, probably through the aza-subtype (13,14), antagonizes the activity of vasopressin (free water clearance) by a mechanism dependent on the circulating catecholamine levels (58). More recently, pharmacological evidence that stimulation of a2A-adrenoceptors increases urine flow has been reported. The mechanism involves an increase in osmolar clearance that can be attenuated by the opioid receptor antagonist naltrexone (13, 14). In conclusion, the present results indicate that a2A-adrenoceptors are upregulated whereas aza-adrenoceptors are not modified in rat kidney following chronic desipramine treatment. The findings are compatible with a differential location and functions of the aradrenoceptor subtypes in the kidney. Acknowledgements This work was supported by grants to J.J.M. from the Spanish CICYT (SAF 9310459) and FIS (95/1731) and by the Basque Government. L.F.C. was supported by the predoctoral training program of the Basque Government. The authors thank the pharmaceutical companies for generous gifts of drugs.

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