202

Brain Research, 517 (1990) 202-208 Elsevier

BRES 15489

Astrocytes cultured from specific brain regions differ in their expression of adrenergic binding sites Paul Ernsberger, Lorraine Iacovitti and Donald J. Reis Division of Neurobiology, Cornell University Medical College, New York, N Y 10021 (U.S.A.) (Accepted 27 October 1989) Key words: fl-Adrenergic receptor; aE-Adrenergic receptor; Astrocyte; Glial cell; Glial fibrillary acidic protein; Neuron-specific enolase; Primary culture; Radioligand binding; p-Aminoclonidine; Dihydroalprenolol We sought to characterize regional heterogeneity ot astrocytes using adrenergic receptor sites as cellular markers. Primary cultures made from 6 regions of neonatal rat brain consisted almost exclusively of astrocytes. Membranes from astrocytes cultured 1-3 weeks were prepared for radioligand binding assays of fl- and a2-adrenergic sites using the ligands [3H]dihydroalprenolol and [3H]p-aminoclonidine, respectively. Receptor expression was not affected by time in culture. Astrocytes from different brain regions varied up to 3-fold with respect to number but not affinity for both classes of adrenergic binding site with a rank order of cerebral cortex = superior colliculus > hippocampus = ventral midbrain-¢audate nucleus~hypothalamus. Binding to fl- and a2-adrenergic receptors was positively correlated across brain regions. Astrocytic receptor binding in each region did not correspond to total receptor levels assessed by quantitative autoradiography. We conclude that: (a) astrocytes are markedly heterogeneous between major brain regions with respect to expression of adrenergic binding sites; (b) regional variations in the density of adrenergic binding sites in brain reflect, in part, local specialization of astrocytes; and (c) a substantial proportion of the adrenergic binding sites in some brain regions may be on astrocytes. INTRODUCTION The distribution of many neurotransmitter receptor binding sites within the brain is m a r k e d l y heterogeneous 15. These regional differences in r e c e p t o r density have traditionally b e e n attributed to local specialization of neurons. A l t h o u g h neurotransmitter receptors are known to be present in astrocytes as well as neurons 16' 18,23

, astrocytes are generally thought to represent a h o m o g e n e o u s p o p u l a t i o n of cells throughout the brain. H o w e v e r , recent evidence indicates that some characteristics of astrocytes vary between different brain regions 11. F o r e x a m p l e , astrocytes in brain areas heavily innervated by g l u t a m a t e fibers exhibit intense immunoreactivity for glutamate d e h y d r o g e n a s e 2 and also show increased glut a m a t e u p t a k e 11. This suggests that astrocytes are not uniform within the brain, and that some astrocytes may participate in n e u r o t r a n s m i t t e r pathways. If astrocytes are h e t e r o g e n e o u s , then they should express non-uniform density of receptors in different brain regions. Conceivably, astrocytes from regions heavily innervated by a particular transmitter might express m o r e receptors than astrocytes from regions lacking innervation. We sought to d e t e r m i n e w h e t h e r astrocytes, like neurons, are regionally specialized with respect to their density of neurotransmitter r e c e p t o r binding sites. To test this possibility, we used tissue culture m e t h o d s to isolate nearly pure

populations of astrocytes from a variety of brain regions and c o m p a r e d their expression of fl- and a2-adrenergic sites. MATERIALS AND METHODS Cell culture Dissociated astrocyte cells were established in culture using a modification of the methods of McCarthy & DeVellis16. Twoday-old rat pups were rinsed in 80% ethanol and decapitated. A variety of brain regions were dissected, including the cerebral cortex, hippocampus, caudate nucleus, ventral midbrain, superior colliculus, and hypothalamus. Individual regions were dissociated in a mild trypsin solution (0.25%), plated into 25 cm2 flasks (Coming) at a density of 4 × 105 ceUs/cm2 and fed Dulbecco's modified medium containing 15% fetal calf serum, 0.1% glutamine, 0.6% glucose, and 1% penicillin/streptomycin. Cultures were maintained in a humidified atmosphere of 95% air and 5% CO 2. At 9-10 days after plating, the cells were shaken on a clinical rotator (200 rpm at 37 °C for 18 h) to remove any possible contaminating neurons and oligodendrocytes. The remaining adherent cells were harvested, diluted, and reseeded at the original plating density into 25 cm2 flasks for binding assays or 4-chamber slides (Lab Tek) for immunocytochemical analysis 1, 2 or 3 weeks later. Time in culture refers to time following replating. lmmunocytochemistry After 1, 2, or 3 weeks in vitro, some cultures were processed for the immunocytochemical localization of either the astrocyte marker, astrocyte fibrillary acidic protein (GFAP), or the neuronal-specific enzyme, neuron-specific enolase (NSE). Cultures were rinsed 3 times in a balanced salt solution, fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4), and incubated with antibodies to either GFAP or NSE at a 1:500 dilution in phosphate-

Correspondence: P. Ernsberger, Division of Neurobiology, Cornell University Medical College, 411 E. 69th St., New York, NY 10021, U.S.A. 0006-8993/90/$03.50 ~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)

203 buffered saline. Antibodies were visualized by peroxidase-antiperoxidase staining using diaminobenzidine as the chromagen21.

i m m u n o c t y o c h e m i c a l l y . N i n e t y - f i v e to 9 9 % o f the cells from a n y b r a i n r e g i o n gave a positive i m m u n o c y t o c h e m -

Membrane preparation

ical r e a c t i o n for G F A P (Fig. 1). Fig. 1 A , B shows typical

Harvested astrocytes were resuspended in medium and counted using a hemocytometer, then centrifuged twice at 500 g for 5 min at 20 °C, with an intermediate resuspension in 25 ml of Earle's balanced salt solution. The pelleted cells were lysed by flashfreezing in a dry-ice acetone bath, and stored at - 70 °C for up to 2 weeks. The lysed cells were slowly thawed, then homogenized in 15 ml of ice-cold sucrose (265 mM) buffered with Hepes (50 mM, pH 7.4) using a teflon-glass homogenizer (5 passes at 1000 rpm). The homogenate was centrifuged at 500 g for 10 min at 4 °C, and the pellet was resuspended in 15 ml HEPES-sucrose and recentrifuged at 500 g. The pooled supernatants were centrifuged at 37,000 g for 15 min, and the pellet was washed twice by centrifugation, once in 50 mM Tris-HCl buffer (pH 7.7) containing 5.0 mM EDTA, and once in EDTA-free Tris-HCl. Flash-frozen pellets were stored at - 70 °C for up to 6 weeks. The protein content of the membrane fractions was determined by the method of Peterson 19. The yield of membrane protein per cell did not vary between brain regions (overall mean of 23 _-+ 1 pg protein/ceil).

results for cultures f r o m the c e r e b r a l cortex a n d the c a u d a t e n u c l e u s , respectively. C u l t u r e s w e r e c o m p o s e d m a i n l y of astrocytes with a flat p o l y g o n a l m o r p h o l o g y b u t s o m e stellate astrocytes were also p r e s e n t . S t a i n i n g for n e u r o n - s p e c i f i c e n o l a s e was o n l y rarely o b s e r v e d in a n occasional cell, e v e n t h o u g h the l a b e l i n g c o n d i t i o n s used result in s t r o n g staining of n e u r o n s in p r i m a r y c u l t u r e la. T h e s e i m m u n o c y t o c h e m i c a l results i n d i c a t e d that these astrocyte cultures c o n t a i n e d few, if a n y , n e u r o n s . T h u s ,

Radioligand binding assays [3H]Dihydroalprenolol ([3H]DHA) binding to fl-adrenergic receptors 9 was assayed by incubating astrocyte membranes (25,000 cells/ml in Tris-HCl) with 1.0 nM [3H]DHA for 20 rnin. Non-specific binding was determined in parallel incubations containing 1 /~M (-)-propranolol. Similar results were obtained in a few experiments using 0.1 mM (-)-isoproterenol to define non-specific binding. [3Hlp-Aminoclonidine ([3H]PAC) binding assays for aE-adrenergic receptors 8 were carried out by incubating membranes (100,000 cells/ml) with 1.0 nM [3H]PAC for 40 min. Guanabenz (10/~M) was used to define non-specific binding because this compound, unlike phentolamine, does not bind to imidazole sites7 which are labeled by [3H]PAC along with a2-adrenergic sites in brain7'8. Astrocyte membranes apparently lack imidazole sites, however, since nonspecific binding defined in the presence of 10/~M guanabenz was not different from that definded by 10/zM phentolamine (135 _+ 42 dpm vs 153 + 45 dpm, n = 7). Furthermore, [3H]PAC binding was not affected by cimetidine (0.1 mM), a potent ligand at imidazole sites7'8 (specific binding = 112 _+ 8% of control, n = 7). Thus, [3H]PAC binds exclusively to a2-adrenergic receptors in glia1°. All incubations were carried out at 25 °C in a final volume of 1 ml and were terminated by vacuum filtration over Whatman GF/B filters using a modified cell harvester (Brandel). The filters were washed with 20 ml ice-cold Tris-HCl, placed in scintillation vials and covered with 5 ml cocktail (Ready-Solv, Beckman) prior to counting at an efficiency of 45%. Saturation data were analyzed by computerized curve-fitting17.

Materials GFAP antibodies were the gift of Dr. D. Dahl, and NSE antibodies were obtained from Polysciences. Both were stored at - 20 °C at a 1:10 dilution in 0.1 M "Iris buffered saline containing 1% goat serum. [3H]PAC (46 Ci/mmoi) and [3H]DHA (82 Ci/mmol) were obtained from New England Nuclear, stored in ethanol at -20 °C, and diluted in reagent-grade water immediately prior to assay. Phentolamine was a gift from Ciba-Giegy. (-)-Propranolol was obtained from Research Biochemicals (Natick, MA) and guanabenz and cimetidine were obtained from Sigma Chemical (St. Louis, MO). RESULTS

Immunocytochemistry of astrocytes cultured from different brain regions T h e cellular c o m p o s i t i o n of the cultures was a n a l y z e d

Fig. 1. Glial fibrillary acid protein (GFAP) reaction product in astrocytes in primary culture. Astrocytes cultured from the cerebral cortex (A) or the caudate (B) of neonatal rat brain were grown 3 weeks after replating and processed for GFAP immunocytochemistry as described in Methods. Note that in both regions all visible cells stain positively. Bar = 15 #m.

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Fig. 3. Scatchard analysis of saturation isotherms in astrocytes cultured from the cortex and the caudate. Astrocyte membranes were incubated with 6 concentrations of (A) [3H]DHA ranging from 0.4 to 16 nM or (B) [3H]PAC ranging from 0.2 to 8 nM. Non-specific binding was defined in the presence of 10 pM guanabenz ([3H]PAC) or 1 pM (-)-propranolol ([3H]DHA) and was a linear function of radioligand concentration and did not differ between regions. Values represent the mean of 3 experiments, each conducted in duplicate. The line of best fit was determined by computerized curve-fitting 17. Astrocytes cultured from neonatal cortex express a greater number of r- and a2-adrenergic binding sites per cell, as indicated by the increased x-intercept (Bmax). However, the apparent affinity of the binding sites for radioligand does not differ between regions, as indicated by the roughly parallel slopes (1/Kd).

205 after 1, 2, or 3 weeks in vitro the cultures consisted of nearly pure populations of astrocytes.

Adrenergic receptor binding in astrocytes from different regions Astrocytic membranes from each brain region showed specific binding of the fl-adrenergic ligand [3H]DHA (Fig. 2A). fl-Adrenergic binding was not uniform, but varied over a 3-fold range. Thus, astrocytes cultured from the cerebral cortex expressed over 3 times as many binding sites for the fl-specific radioligand [3H]DHA as did hypothalamic astrocytes. The rank order of binding was (in descending order): cerebral cortex -- superior colliculus > hippocampus = ventral midbrain = caudate nucleus > hypothalamus. A similar range was obtained when data were corrected for protein rather than expressed as sites per cell (cerebral cortex: 56 _+ 6 fmol/mg protein; hypothalamus: 22 + 8 fmol/mg protein). EFFECT OF TIME IN CULTUREON EXPRESSIONOF ADRENERGIC RECEPTORS 300 -

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a2-Adrenergic binding for astrocytes from each region is shown in Fig. 2B. In descending order: superior colliculus -- cerebral cortex > ventral midbrain = hippocampus > hypothalamus = caudate nucleus. The nearly 3-fold difference in binding sites per cell b e t w e e n superior colliculus and hypothalamus persisted when the data were corrected for protein (superior colliculus: 12 _+ 2 fmol/mg protein; caudate: 3.4 + 0.2 fmol/mg protein). There was a positive correlation between a 2- and fladrenergic binding in astrocytes from different regions (r = 0.95, n = 6, P < 0.05), indicating that regions with a high density of one subtype of adrenergic receptor site tended also to express high levels of the other subtype. To determine whether the distribution of astrocyte adrenergic receptors is independent of the distribution of neuronal receptor binding sites, the 6 brain regions were ranked according to relative receptor density in whole tissue of adult rat brain by receptor autoradiography as reported in previous studies 2°'25. The estimated rank order for fl-receptor density in brain tissue was: caudate nucleus > cerebral cortex > superior colliculus > hippocampus > ventral midbrain > hypothalamus 2°. This ranking was unrelated to astrocyte fl-receptor binding (rank-order correlation = 0.43). The ranking for a 2adrenergic sites was: hippocampus > cerebral cortex > caudate nucleus > hypothalamus > superior coUiculus > ventral midbrain 25. The densities of aE-receptor sites in astrocytes and whole tissue were unrelated (rank-order correlation -- - 0.26).

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receptors. Specific [3H]DHA binding to fl-adrenergic sites (light bars, scale at left) and [3H]PAC binding to a2-adrenergic sites (dark bars, scale at right) were assayed in astrocytes grown for different lengths of time in culture. Identical aliquots of astrocyte cell suspension were plated in 3 dishes, which were harvested after 1, 2 or 3 weeks of culture. Time in culture had no significant effect on [3H]DHA or [3H]PAC binding.

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In order to determine whether regional differences in receptor binding reflected changes in radioligand affinity rather than actual variations in the number of binding sites per cell, saturation binding curves were obtained for astrocytes from 2 representative brain regions. Astrocyte membranes from the cerebral cortex or the caudate nucleus were incubated with increasing concentrations of [3H]DHA or [3H]PAC. Scatchard plots of the resulting saturation isotherms, shown in Fig. 3, revealed that the number of fl- and a2-adrenergic binding sites per cell (Bmax) was significantly greater in cerebral cortex than in the caudate nucleus. When corrected for membrane protein, the difference in fl-adrenergic density between astrocyte membranes from the cerebral cortex and the caudate nucleus persisted (64 + 8 vs 22 + 4 frnol/mg protein). The density of fl-adrenergic sites in astrocytes from cerebral cortex agrees closely with that previously reported using [125I]iodocyanopindolol as a ligand 24. The specific concentration of a2-adrenergic sites was also greater in cerebral cortical astrocytes than in caudate nucleus astrocytes (16 _ 2 vs 9 + 1 fmol/mg protein).

206 Radioligand affinity did not differ between the 2 areas, as indicated by the parallel slopes of the Scatchard plots. The affinity of [3H]PAC and [3H]DHA for astrocyte membrane sites closely agrees with previous values obtained in whole brain tissue using similar methods 8"9.

Effects of time in culture on adrenergic receptor site expression in astrocytes The expression of adrenergic binding sites by astrocytes may be affected by culturing, and thus regional variations in receptor binding may reflect a differential response to the culture environment rather than a difference present in vivo. We therefore harvested cerebral cortical astrocytes after 1, 2, or 3 weeks in culture. As shown in Fig. 4, fl- and a2-adrenergic binding was not affected by length of time in culture, suggesting that the present culture conditions do not lead to either induction or suppression of adrenergic receptor expression in astrocytes. DISCUSSION The present study demonstrates that astroglia removed from regions of neonatal rat brain and grown in the absence of neurons for up to 3 weeks in primary culture express fl- and a2-adrenergic binding sites at a stable high level. Moreover, we found that the number but not affinity of adrenergic sites on astrocytes varies regionally over a 3-fold range, suggesting that astrocytes may be locally specialized with respect to adrenergic receptor density. It is unlikely that the regional variations in astroglial adrenergic binding were an artifact of the culture conditions. Expression of both adrenergic receptor subtypes on astrocytes was stable up to 3 weeks in culture, suggesting that levels of receptor expression in vitro approximate neonatal levels in vivo. Similarly, autoradiographic studies by others 23 have demonstrated that the density of fl-adrenergic sites on individual polygonal astrocytes were also stable up to 3 weeks in vitro. In contrast, another study reported that fl-adrenergic stimulation of adenylate cyclase in cultured cerebral cortical astrocytes increased 9-fold between the first and second week in culture, then decreased 3-fold by the third week H. These dramatic effects of time in culture, which were not observed by us or by others 23, may result from differences in culture conditions. The failure of astroglial adrenergic ligand binding sites to increase over 3 weeks in culture is of interest. During early postnatal development fl- and a2-adrenergic sites increase several-fold throughout the brain 4'22. This difference between the maturation of receptors in vitro and in vivo could indicate that the early postnatal induction

of adrenergic binding sites may be specifically neuronal. Alternatively, astrocytes may contribute to the postnatal rise in the number of adrenergic binding sites in brain, but the factors that induce adrenergic receptor expression in vivo are lacking in cell culture. The present study confirms and extends previous studies demonstrating the existence of fl- and a2-adrenergic receptors in astrocyte cells from the cerebral cortex in primary culture 16'18'24. Furthermore, we report that adrenergic binding sites are present in astrocytes cultured from regions other than the cerebral cortex. The binding properties of adrenergic binding sites on cultured astrocytes of the cerebral cortex and the caudate nucleus are nearly identical to those observed in brain tissue under similar assay conditions 4'9, suggesting that adrenergic receptor proteins expressed by astrocytes and neurons are the same. Indeed, a specific antibody directed against a restricted sequence of the fl-receptor labels both neurons and astrocytes 1'3. The cellular mechanism accounting for regional variations in astrocytic adrenergic binding sites is not certain. One possibility is that local populations of astrocytes within any given brain region express a characteristic level of binding site density. Alternatively, regional heterogeneity might reflect the differential distribution of astrocytic subtypes differing in adrenergic binding site density. In support of the latter hypothesis, autoradiographic studies demonstrate that polygonal or Type I astrocytes contain a higher density of fl-adrenergic receptors than fibrous or Type II astrocytes23. In our own preliminary studies we have used a photoaffinity ligand to covalently label fl-adrenergic binding sites on intact astrocytes, allowing combined localization of receptors and immunocytochemical markers on individual cells5. We have observed that the differences in fl-adrenergic receptor binding between astrocytes derived from the cerebral cortex and the caudate nucleus appears attributable to variations in adrenergic binding site density on individual cells. Although the present study is the first to demonstrate regional differences in fl- or a2-adrenergic receptor binding in astrocytes, fl-receptor function has been assessed by Hansson 11 in astrocytes derived from 3 different brain regions of neonatal (7-day-old) rat pups. After 3 weeks in culture, the rank order of potency for fl-adrenergic stimulation of adenylate cyclase was: cerebral cortex > > brainstem > striatum. In the present study, we obtained a comparable order for fl-receptor binding in astrocyte membranes: cerebral cortex > > midbrain > caudate. Thus, the regional differences in the number of fl-adrenergic binding sites on astrocytes probably represent differences in functional fl-receptors coupled to adenylate cyclase.

207 Regional variations in the number of adrenergic binding sites on astrocytes do not appear to correlate with neuronal binding site density. Astrocytes cultured from brain regions containing high densities of adrenergic sites by quantitative autoradiography 2°'25 showed no tendency to exhibit higher levels of binding. Thus, the distribution of astrocyte receptors appears to be independent of the distribution of neuronal receptors. Interestingly, the densities of r - and a2-adrenergic binding sites in different regions were positively correlated, suggesting that if there is a region-specific factor inducing expression of astrocyte adrenergic sites, it affects both subtypes. Regional density of astrocytic binding sites may be related to noradrenergic innervation. The cerebral cortex, which is heavily innervated, contained higher densities of r - and a2-adrenergic sites than the caudate nucleus, which is sparsely innervated. It is thus conceivable that the expression of fl-adrenergic sites by astrocytes is enhanced in the vicinity of noradrenergic synapses. This possibility is supported by recent ultrastructural evidence for high densities of immunoreactive fl-adrenergic receptors on glia lying near terminals containing tyrosine hydroxylase immunoreactivity3 or on glia adjacent to neuronal postsynaptic densities containing fl-adrenergic receptor immunoreactivity ~. Surprisingly, astrocytes from the hypothalamus show very low levels of adrenergic binding sites despite heavy noradrenergic innervation. This suggests a complex relationship between astrocytic expression of receptors and local concentrations of endogenous ligand. In adult brain tissue, the regional distribution of neurotransmitters often 13 but not always 6 fails to coincide with the localization of the relevant receptors. One of the most striking examples of so-called transmitter-receptor mismatch is the remarkably high density of fl-adrenergic binding sites in the caudate nucleus, which is only lightly innervated by noradrenergic fibers ~3. Previous radioligand binding studies of fl-adrenergic sites in the caudate nucleus have not distinguished between glial and neuro-

nal receptors. The present study indicates that astrocytes from the caudate nucleus express relatively low levels of adrenergic sites (see Figs. 2 and 3). Thus, the high density of fl-adrenergic sites in the caudate nucleus is apparently localized to neurons. The demonstration of regional variations in astrocytic binding sites has relevance to the interpretation of binding site distributions throughout the brain, since the contribution of glial sites is not trivial. Direct comparisons between our results and previous data obtained from slide-mounted sections or homogenates of brain tissue 9' 2o,25 are difficult. However, if we estimate that astrocytes contribute 'approximately 40% of the total membrane protein in brain 12 (M.E. Hatten, personal communication), we can estimate that astrocytes contribute at least one-third of the total population of fl-adrenergic binding sites in cerebral cortex, one-eighth in the caudate nucleus, and one-fourth in the hypothalamus. Astrocytic a2-sites make up as little as one-tenth of the population of these binding sites in the hippocampus. In contrast, high concentrations of aE-adrenergic binding in astrocytes from the superior coiliculus were associated with a low tissue content of sites, suggesting that the majority of the binding sites here are non-neuronal. In conclusion, regional variations in binding site density in whole tissue may reflect local specialization of astrocytes as well as neurons. Anatomical mapping of binding sites by quantitative autoradiography 5'6'15 may be influenced by variations in astrocyte as well as neuronal function. This conclusion is strengthened by ultrastructural evidence 1'3. Future studies of the distribution of binding sites in brain should take into account the contribution of astrocytes.

Acknowledgements. This work was supported in part by NIH Grant HL18974. We gratefully acknowledge Ms. Liubov Lyandvert for expert assistance in tissue culture and Mr. Gary Feinland for conducting radioligand binding assays. We thank D. Dahl for providing anti-GFAP antiserum.

REFERENCES 1 Aoki, C., Joh, T.H. and Pickel, V.M., Ultrastructural localization of fl-adrenergic receptor-like immunoreactivity in the cortex and neostriatum of rat brain, Brain Research, 437 (1987) 264-282. 2 Aoki, C., Milner, T.A., Sheu, K.-ER., Blass, J.P. and Pickel, V.M., Regional distribution of astrocytes with intense immunoreactivity for glutamate dehydrogenase in rat brain: implications for neuron-astrocyte interactions in glutamate transmission, J. Neurobiol., 7 (1987) 2214-2231. 3 Aoki, C., Zemik, B.A., Strader, C.D. and Pickel, V.M., Cytoplasmic loop of fl-adrenergic receptors: synaptic and intracellular localization and relation to catecholaminergic neurons in the nuclei of the solitary tracts, Brain Research, 493 (1989) 331-347. 4 Bylund, D.B. and U'Prichard, D.C., Characterization of a t-

5

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and a2-adrenergic receptors, Int. Rev. Neurobiol., 24 (1983) 343-431. Ernsberger, P., Arango, V., Iacovini, L. and Reis, D.J., Photoaffinity labeling of benzodiazepine receptors in slidemounted sections and fl-adrenergic receptors in intact glial cells, Soc. Neurosci. Abstr., 14 (1988) 170. Ernsberger, P., Arneric, S.P., Arango, V. and Reis, D.J., Quantitative distribution of muscarinic receptors and choline acetyltransferase in rat medulla: examination of transmitterreceptor mismatch, Brain Research, 452 (1988) 336-344. Ernsberger, P., Giuliano, R., Willette, R.N., Granata, A.R. and Reis, D.J., Hypotensive action of clonidine analogs correlates with binding affinity at imidazole and not a2-adrenergic receptors in the rostral ventrolateral medulla, J. Hypertens., 6, Suppl. 4 (1988) $554-$557. Ernsberger, P., Meeley, M.P., Mann, J.J. and Reis, D.J., Clonidine binds to imidazole binding sites as well as ct2-

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