Binding characteristics of the selective α2-adrenoceptor antagonist [3H]idazoxan to rat olfactory cortex membranes

European Journal of Pharmacology, 121 (1986) 91-96

91

Elsevier

BINDING CHARACTERISTICS OF THE SELECTIVE a2-ADRENOCEPTOR ANTAGONIST [3HIIDAZOXAN TO RAT OLFACTORY CORTEX MEMBRANES N.J. MCLAUGHLIN * and G.G.S. COLLINS : Department of Pharmacology, University of Sheffteld, Western Bank, Sheffield S IO 2TN. U.K.

Received 30 May 1985, revised MS received 23 October 1985, accepted 5 November 1985

N.J. MCLAUGHLIN and G.G.S. COLLINS, Binding characteristics of the selectioe a,-adrenoceptor antagonist [3H]idazoxan to rat olfactory cortex membranes, European J. Pharmacol. 121 (1986) 91-96. [3H]Idazoxan binding to membranes prepared from rat olfactory cortex obeyed saturation kinetics and was to a single population of sites. Although the density of sites was dependent on the incubation medium, binding was of high affinity (K D approximately 5.5 nM) with a Hill coefficient close to unity. Competition studies with a range of adrenoceptor agonists and antagonists confirmed that [3H]idazoxan binding was to a2-adrenoceptors. Neither chemical lesions with the neurotoxin kainic acid nor chronic unilateral bulbectomy significantly altered any of the [3 H]idazoxan binding parameters. These findings suggest that a2-adrenoceptors are not located on the lateral olfactory tract terminals or pyramidal cells of the olfactory cortex. Olfactory cortex

[ 3H]ldazoxan

a2-Adrenoceptors

1. Introduction There seems little doubt that more than one type of a-adrenoceptor exists in mammalian tissues (Langer, 1981; Starke, 1981). In the central nervous system, ligand binding techniques have provided direct evidence for the presence of a zadrenoceptors in a number of brain regions. Included amongst the radio-labelled ligands employed in such studies is idazoxan (RX 781094; [2-(2-(l,4-benzodioxanyl)-2-imidazoline]), a potent a2-adrenoceptor antagonist which possesses central nervous system activity and a greater a2-adrenoceptor selectivity than either rauwolscine or yohimbine (Doxey et al., 1982; 1983a,b; Dettmar et al., 1981; Howlett et al., 1982; Lane et al., 1983; Langer et al., 1983). Application of low concentrations of noradrenaline to olfactory cortex slices can cause both a * To whom all correspondence should be addressed. * Present address: Department of Biological Sciences, Manchester Polytechnic, All Saints, Manchester M15 6BH, U.K. 0014-2999/86/$03.50 ~3 1986 Elsevier Science Publishers B.V.

Kainic acid

Bulbectomy

potentiation and inhibition of excitatory transmission at the lateral olfactory tract-pyramidal cell synapse (Collins et al., 1984), effects which are antagonized by non-selective a- a n d / 3 - a d r e n o c e p tor antagonists. The present paper describes the results of some experiments in which the binding of [3H]idazoxan to olfactory cortex membranes was measured. The aim was directly to confirm the presence of a2-adrenoceptors in the olfdctory cortex and by chemical and surgical lesioning techniques, to identify their cellular location(s).

2. Materials and methods Male albino rats (180-220 g) of a Wistar strain were used in all experiments. [3H]Idazoxan (23.5 C i / m m o l with a stated purity > 99%) was the kind gift of Dr. J.C. Doxey, Reckitt and C o l m a n Ltd., U.K.). All radiochemicals required for the microdansylation assay of amino acids (see Clark and Collins, 1976) were purchased from Amersham International, U.K. The following compounds were gifts from the companies indicated:

92 chlorpromazine and thioproperazine, May and Baker Ltd., U.K.: clonidine. Boehringer lngelheim, F.R.G.: labetolol, Giaxo Research Ltd., U.K.: phentolamine and tolazoline, CIBA Laboratories, U.K. All other drugs were purchased from Sigma, U.K.

2.1. Preparation of membranes Olfactory cortex slices were prepared using a guide recessed to 0.5 mm and a bow cutter and immediately homogenized using a glass-Teflon homogenizer in 20 volumes of ice-cold buffer (Tris/HCl, 50 mM, pH 7.7) containing 0.2% ascorbate. The homogenate was centrifuged at 50000 × g for 10 min at 4°C, the pellet resuspended in buffer and then centrifuged for a second time. The final pellet was resuspended in 100 volumes of either the buffer or a physiological salt solution (composition in mM: NaCI 118.1, CaCI 2 2.5, MgSO4 1.1, KCI 2.1, KH,PO4 0.93, NaHCO3 25, glucose 11, ascorbate 0.1) equilibrated to pH 7.3-7.4 by gassing with 5% CO2 in O~.

2.2. Lesioning experiments In the first series of experiments, rats were injected i.p. with 12.5 m g / k g of the neurotoxin kainic acid. Three to six days later, the animals were killed and olfactory cortex slices cut in the usual manner. A control group of animals was injected with an equivalent volume of sabine. In most experiments, 20-30 slices were pooled and membranes prepared using the procedure described in 2.1. In order to check the neurotoxic effectiveness of the drug, amino acids were extracted from some of the slices and the contents of endogenous aspartate, glutamate and GABA measured using a sensitive double label microdansylation procedure (for full detials, see Clark and Collins, 1976). In the second series of experiments, rats were unilaterally bulbectomized under pentobarbitone anaesthesia (Collins, 1979b) and sacrificed 4-6 days following surgery.

2.3. Binding experiments All binding experiments were carried out using freshly prepared membranes and the incubations

were in triplicate. The incubation mixture contained membrane suspension (1 000 p,l containing 0.2-0.3 mg protein), saturating or competing ligand (200 ~1) and [~H]idazoxan (40 ~1 to give a final concentration between 0.25 and 15 nM) made up in either Tris/HC1 buffer (50 raM, pH 7.7) or physiological salt solution to givc a final volume of 2000 ~1. Specific [3H]idazoxan binding was defined by the addition of phentolamine (20 #M). Samples were incubated for 30 min at 25°C and then rapidly filtered under reduced pressure through Whatman G F / B glass filters. The filters were washed once with 10 ml ice-cold Tris/HCI buffer (50 mM, pH 7.7), dried in an oven and the radioactivity estimated in a Packard 460 CD scintillation spectrometer using a proprietary counting cocktail (Scintran-T, British Drug Houses). The K17 and B...... (fmol/mg protein) values were determined from Scatchard analysis and Hanes plots. Protein was measured (Lowry et al., 1951) using bovine serum albumin as standard. Competition binding experiments were carried out using a single concentration of [3H]idazoxan (1 nM) and up to 5 concentrations of each competing ligand. Pseudo Hill plots were drawn for each competing ligand: these were linear regressions of log ( B - B I ) / B t versus log ligand concentration where B and B~ represent the specific binding of [3H]idazoxan alone and in the presence of a given concentration of agonist/antagonist respectively. The concentration of each ligand which inhibited specific [ ~H]idazoxan binding by 50% (IC~, values) was calculated. In addition, inhibition constants (K, values) were estimated using the method described by Cheng and Prussoff (1973).

3. Results

The binding of ligands to a2-adrenoceptors is dependent on the composition of the buffer used in the incubations (Rouot et al., 1982: Glossman and Presek, 1979). With [ 3H]idazoxan, physiological salt solutions have been reported to give satisfactory binding characteristics (Doxey et al., 1983a; Lane et al., 1983) and so most of the binding experiments reported in the present paper were carried out using salt solutions. However, both salt

93 solutions a n d Tris-HCl buffer (50 mM, pH 7.7) were used in the studies of the kinetics of [~ H]idazoxan binding.

3.1. Specific [3H]idazoxan binding At a c o n c e n t r a t i o n of [3H]idazoxan of 1 nM, specific b i n d i n g constituted 75-80% of the total a n d was linear over a protein c o n c e n t r a t i o n range up to 0.5 m g / s a m p l e and an i n c u b a t i o n time of at least 60 m i n (not shown). The temperature-dep e n d e n c y of b i n d i n g was not investigated. In experiments carried out in physiological salt solution, b i n d i n g of [3H]idazoxan obeyed saturation kinetics over the c o n c e n t r a t i o n range tested (0.5-15 nM). Scatchard a n d Hanes analysis of the results

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Log [3HI Idazoxan --

.

Values of Bmax, K o and Hill coefficients for the specific binding of [3H]idazoxan to membranes prepared from rat olfactory cortex. Results are means 5-S.E.M. of 6 experiments. Bm~Xand K i) were determined by Scatchard analysis of saturation i~therms. Incubation medium

Bm~~ Ko (fmol/mg (nM) protein)

Hill coefficient

Physiological salt solution 778 + 54 * 5.58 5-0.45 1.04 5- 0.l)1 Tris/HCl buffer (50 mM, pH 7.7) 251 5- 8 * 5.71 -t-0.42 1.04 + 0.01 * Significantlydifferent (Student's t-test, P < 0.001). showed that b i n d i n g was to a single class of sites (r = 0.99) (see fig. 1). The mean values of K D, B.... a n d Hill coefficients for b i n d i n g are given in table 1. W h e n the experiments were repeated in Tris-HCl buffer, the Bma, value was considerably less although there were no differences in the K t) values a n d Hill coefficients (table 1).

3.2. Effects of drugs on the specific binding of [ 3H]idazoxan

"x

[3HI Idazoxan (nM) +0.5

TABLE 1

X IO"M

Fig. 1. Specific binding of [3H]idazoxan to olfactory cortex membranes measured in physiologicalsalt solution. The data is from a typical experiment and each point is the mean of a triplicate measurement. (A) Saturation binding curve with a Scatchard plot of the data shown in the inset. The line of the plot is a linear regression determined by least squares fit (r = - 0.98) and gave a calculated K t) of 5.29 nM and a Bmax of 753 fmol/mg protein. Mean values are given in table 1. (B) Hill plot of the same data (r = 0.99); the slope of the line is + 1.01 and mean values are given in table 1.

The effects of a range of adrenoceptor agonists a n d antagonists on the specific b i n d i n g of [3H]idazoxan (1 n M ) was tested in order to identify the sites to which the labelled ligand was b i n d i n g (fig. 2 a n d table 2). ldazoxan, y o h i m b i n e a n d clonidine were some of the more potent displacers whereas prazosin was very much less active. With the exception of clonidine, the a - a d r e n oceptor agonists were all less potent displacers of [3H]idazoxan b i n d i n g than were the antagonists. D o p a m i n e , cocaine a n d the neuroleptics chlorp r o m a z i n e a n d thioproperazine showed little affinity for the idazoxan b i n d i n g site (table 1). The Hill coefficient for unlabelled idazoxan was not significantly different from unity although there was a tendency for the coefficients of the agonists to be lower than those of the antagonists (table 2).

3.3. Lesioning experiments Approximately 2 h following the i.p. injection of kainic acid (12.5 m g / k g ) into rats a characteris-

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"I ABI.E 3 Effect of chemical and surgical lesions on the ",alues of Bm,,x. Kt~ and 11ill coefficients of specific [aHlidazoxan binding. Values are means~ S.E.M. Full experimental details arc given m the text. In these experiments, incubations were carried m physiological sah solution.

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"I'ABLI:~ 2 Inhibition of [aH]idazoxan binding by various drugs. Each drug was tested at between 4 and 7 concentrations. The l('s~~ values and pseudo-Hill coefficients in u) were determined from Hill plots and the apparent K, values calculated from K, = l('>,,/il +[labelled ligand]/Kt~) (('heng and Prussoff, 19731. K, (nM)

nu

n

1.6_+ 0.09 11.3 f 1.1 22.7 z 3.7 87.5.4 19.6 748 + 5 1 772 ± 223 > 30000

1.113_+0.03 0.84 f 0.08 0.84_+0.03 0.87 _+0.(}4 0.92_+0.03 0.85 -I.~/).05

5 4 4 4 6 4 3

0.90+(I.03 0.58_+0.04 0.95_+0.03 0.83+0.02 0.58_+0.02 0.61 +0.04

4 6 4 4 4 4

a-Antagonists ldazoxan Phentolamine Yohimbine lolazoline Prazosin Phenoxybcnzaminc l.abctolol cx-Ag,onists ('lonidmc / )Adrenaline all-Amphetamine t.-Phenylephrine (2)Noradrenaline Methoxamine Others Chl,.wpronlazine lhiopr,.~perazine l)opamine

Cocaine

55.9± 1238 -1399 _+ l 534 + 2883 e 5767 ±

5.8 173 130 125 315 41(I

1713 _+ 282 2281 + 376 14671 ±1295

> 30000

K x~ (nM)

Hill coefficient

737 2 61

5.36 + 0.50

1.(13+ (I.01

710 4- 40

5.72 + 0.24

1.04± (I.01

743.+_64 71)2.+ 45

6.18+0.62 6.06 ,'- 0.31

1.03+0.(11 1.04 + 0.01

( "hertll{tg/leslotlA"

(M)

I"ig. 2. Displacement of [3H]idazoxan binding by the adrenoccptor antagonists idazoxan (1). phentolarnine 12), yohimbine (31, tolazolinc (4), prazosin (5) and phenoxybenzamine i6). The results are illustrated in the form of Hill plots showing linear regressions of log (B- B t )/B 1 against antagonist concentrations where the specific [3H]idazoxan binding alone and in the presence of a given antagonist concentration is represented by B and B I respectively. Each point is the mean of at least 4 determinations done in triplicate. Values of the Hill coefficients are given in table 2.

Drug

B...... (fmol/mg protein)

0.98±0.05 3 0.81 +_0.04 3 0.84 .~.0.05 3

3

('ontrol (n = 61 isaline i.p.) Kainic acid in = 41 ( 12.5 mg/kg i.p.) Surgical lesions ('ontrol side in = 41 Bulbectomized side (n = 4)

tic s y n d r o m e a p p e a r e d w h i c h i n c l u d e d w e t d o g shakes, masticatory movements, rearing, forelimb chmus, proptosis and occasional generalised s e i z u r e s (2 o u t of 25 a n i m a l s ) . F o u r d a y s f o l l o w i n g t r e a t m e n t , t h e r e w a s a s i g n i f i c a n t ( S t u d e n t ' s t-test, P < 0.05) r e d u c t i o n in t h e a s p a r t a t e a n d g l u t a m a t e c o n t e n t s o f o l f a c t o r y c o r t e x slices f r o m 4.3 + 0.5 and 8.0+0.3 to 2 . 5 + 0 . 2 and 4.7_+0.8 respectively ( m e a n v a l u e s in p, m o l / g wet w e i g h t + S . E . M . , n = 5) w h e r e a s g l u t a m i n e a n d G A B A levels w e r e u n a l t e r e d ( n o t s h o w n ) . A l t h o u g h t h e B...... o f binding was reduced after kainate treatment, the effect was not statistically significant and the Kt) v a l u e s a n d Hill c o e f f i c i e n t s w e r e u n a f f e c t e d ( t a b l e 3). S i m i l a r l y , t h e b i n d i n g o f [ 3 H ] i d a z o x a n to o l f a c tory cortex membranes from bulbectomized rats w a s n o t s i g n i f i c a n t l y a f f e c t e d ( t a b l e 3).

4. Discussion The present experimental results show that [ 3 H ] i d a z o x a n b i n d s to o l f a c t o r y c o r t e x m e m b r a n e s with high affinity and that the binding obeys s a t u r a t i o n k i n e t i c s . T h e d e n s i t y o f b i n d i n g sites m e a s u r e d u s i n g T r i s - H C I b u f f e r in t h e i n c u b a t i o n s (251 f m o l / m g p r o t e i n ) c o m p a r e s f a v o u r a b l y w i t h v a l u e s r e p o r t e d f o r t h e w h o l e c o r t e x ( H o w l e t t et al., 1982; L a n g e r et al., 1983: L a n e et al., 1983,

95

Doxey et al., 1983a). However, the apparent density of binding sites was increased approximately 3-fold when incubations were carried out in physiological salt solution (table 1). There is no evidence that this increase reflects binding to other sites (for example, sites concerned with uptake) as the displacement of [3H]idazoxan binding by a range of drugs gave no indication of binding heterogeneity (fig. 2 and table 2). Although the binding of ligands to a2-adrenoceptors shows cationdependency (Rouot et al., 1982; Woodcock and Murley, 1982), Lane et al. (1983) reported that binding of [3 H]idazoxan measured in physiological salt solution and Tris-HC1 buffer was similar. The binding of [3 H]idazoxan to olfactory cortex membranes was to a single population of sites which possessed the characteristics of az-adrenoceptors. Thus in the competition experiments, az-selective agonists such as clonidine had higher affinities for the idazoxan binding site than did the a~-selective agonists phenylephrine and methoxamine. Similarly, the a:-selective antagonists idazoxan and yohimbine were very much more potent displacers of [3H]idazoxan binding than was the al-selective antagonist prazosin. Antagonists of/3-adrenoceptors were almost inactive in preventing specific [3H]idazoxan binding (not shown) although the neuroleptic drugs chlorpromazine and thioproperazine, which in addition to blocking dopamine receptors also show some affinity for a~-adrenoceptors (Huerta-Bahena et al., 1983), did partially inhibit binding. These results show that a2-adrenoceptors are indeed present in the olfactory cortex and are consistent with their autoradiographic identification in the anterior pyriform cortex (Young and Kuhar, 1980). The lesioning experiments were undertaken to determine the effects of the loss of specific cell types on [3H]idazoxan binding and hence to identify the location of the a2-adrenoceptors. The olfactory cortex is extremely sensitive to the neurotoxic actions of kainic acid (Heggli et al., 1981), characteristically showing a profound loss of pyramidal cells following either local or i.p. administration of kainate (Olney and De Gubareff, 1978). The loss of both aspartate and glutamate following kainate treatment in the present experiments is consistent with those earlier findings for

both excitatory amino acids have been proposed as pyramidal cell transmitters in the olfactory cortex (Collins et al., 1981). The failure of kainic acid to affect [3H]idazoxan binding suggests that the ~:adrenoceptors of the olfactory cortex are not located on the pyramidal cells. Surgical bulbectomy leads to a rapid degeneration of the lateral olfactory tract terminals (Heimer, 1968) which is followed by a transneuronal degeneration of some of the pyramidal cells (Heimer and Kalil, 1978). In the rat, bulbectomy leads to a loss of aspartate and glutamate in the olfactory cortex (Harvey et al., 1975; Godfrey et al., 1978; Collins, 1979a; 1984). However, bulbectomy also failed to alter the binding of [3 H]idazoxan suggesting that the a2-adrenoceptors of the olfactory cortex are not located on the lateral olfactory tract terminals. In conclusion, the characteristics of [3 H]idazoxan binding are strongly indicative of the presence of a2-adrenoceptors in the rat olfactory cortex. However, they appear not to be located either on the lateral olfactory tract terminals or pyramidal cells. A primary site of action of noradrenaline in modifying synaptic transmission in the olfactory cortex is thought to be the tract terminals (Collins et al., 1984): if this is so, then the effects of noradrenaline cannot be mediated by c~,-adrenoceptors.

Acknowledgement This work was supported by the Medical Research Council IU.K.) by a grant to G.G.S.C.

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