Glial and neuronal opioid receptors: apparent positive cooperativity observed in intact cultured cells

Brain Research. 441 (1988)41-47 Elsevier

41

BRE 13265

Glial and neuronal opioid receptors: apparent positive cooperativity observed in intact cultured cells Katalin Maderspach and Revaz Solomonia* b~stitute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged (Hungary)

(Accepted 28 July 1987) Key words: Opioid receptor: Naloxone; Opioid agonist; Glia; Neuron; Brain culture

Opioid receptors were characterized in glial and neuronal homogeneous cultures of embryonic chick forebrain, using [3H]naloxone as a labelled ligand. Binding experiments were performed on intact cells. The specific binding of [3H]naloxone reached equilibrium after 1 min. The apparent dissociation constants were estimated as 0.51 nM for glial and 0.63 nM for neuronal cells. Equilibrium measurements indicated the apparent positive cooperativity of the binding, resulting in Hill coefficients of 2.61 for glial and 2.04 for neuronal cells. Competition of unlabelled naloxone for specific binding sites resulted in maximum-shapecurves in glial cells if measured at low receptor occupancy. This supports the positive cooperativity of ligand binding. Opioid agonists, ethylketocyclazocine (EKC), morphine and [D-Ala",L-LeuSlenkephalin(DALA), provoked biphasic competition curves in both cell types with a characteristic maximum at low competitor concentrations. The possible physiological role of glial opioid receptors in neuroa-glia communication and the significanceof cooperativity is discussed. INTRODUCTION Opioid receptors appear in the brain at a very early stage of embryonic life. Gibson et al.II characterized receptors by the binding of [3H]etorphine in 4-dayold chick embryos. Different subtypes of the opioid receptors were present in mouse embryonic brain, at day 15 (ref. 28). Opioid receptors can be detected in cell cultures derived from the embryonic nervous tissue. Lenoir et al. 16 found stereospecific [3H]dihydromorphine binding in foetal rat brain aggregate cultures, and the down-regulation of the receptors by etorphine was also shown. Studying chick brain neuron-glia cultures, Peterson et ai. 23 demonstrated, that morphine sulphate (2.5 x 10-4 M) increases the choline acetyltransferase and acetylcholinesterase activities of neurons. These cells exhibited the phenomenon of tolerance. Recently Sakellaridis et al. 25 measured the effect of morphine (10 -5 M) on neurons growing on poly-L-lysine and reported a neurotoxic

effect coupled to a dramatic decrease in choline acetyltransferase actixity. Morphine also promoted the development of glial cells. These authors supposed, that neuronai-glial interactions influence the response of neurons to opiates. This seems to be possible, because in the above experiment 23 the glial cells protected the neurons from the toxic effect of morphine. Hendrickson and Lin 12 characterized opioid receptors by the [3H]naloxone binding in chicken brain homotypic neuronal aggregate cultures. Glial aggregate cultures, however, failed to present stereospecific ligand binding. The authors have drawn the conclusion that opioid receptors are present only i~a neuronal cells. Some data, however, seems to oppose this opinion. Albouz et al. 1 found stereospecific [3H]dihydromorphine binding in C6 glioma cells after desipramine-provoked lipid modifications and supposed opioid receptors also on normal glial cells. Rougon et al. 24 as well as Pearce et al. 22 confirmed the presence of opioid receptors on glial cells (rat

* Present address: Department of Neurochemistry, I.S. Beritashvili Institute of Physiology, Georgian Academy of Sciences, 14, L. Gotua Street, Tbilisi 380060, U.S.S.R. Correspondence: K. Maderspach, Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 521,6701 Szeged, Hungary. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

42 astrocytes) on the basis of the results that both morphine and methionine-enkephalin modified the effect of noradrenaline to glycogen metabolism. The characterization of glial opioid receptors by the ligand-binding assay was not established, however, until recently. In the present work we measured glial opioid receptors parallel to neuronal ones in homogenous primary cultures of the chick embryonic brain 17"~s [3H]Naloxone binding experiments were performed on intact, living cells. This was of significance because homogenization may strongly influence the ligand-binding characteristics of the transmitter receptors7.17.30.31 MATERIALSAND METHODS White Leghorn chick embryos were obtained from a local poultry farm. Chemicals used for cell cultivation were from Gibco; other chemicals were from Sigma. Plastic dishes (6 cm diameter) were made by Falcon. [3H]Naloxone (spec. radioact. 3.1 TBq/ m m o l = 83.7 Ci/mmol) was prepared by Dr. G. Toth -'7. Naloxone hydrochloride was kindly donated by Endo Laboratories. Dextrorphan tartrate was kindly provided by Hoffman-LaRoche. [D-Ala2,LLeu'~]enkephalin (DALA) was synthesized by K. Medzihradszky and coworkers (Central Research Institute for Chemistry, Budapest, Hungary). Ethylketocyclazocine (EKC) was donated by Sterling Winthrop Research Institute. All other chemicals were of analytical grade.

Cultivation procedures Chick embryonic forebrains were mechanically dissociated by a procedure given originally by Booher and Sensenbrenner 4. Eagle MEM, supplemented with 20% foetal calf serum and antibiotics was used as a growth medium. Neuronal cultures were obtained from 7-day-old embryos, seeding the cell suspension into Petri dishes covered previously with poly-L-lysine -'6 at an average density of l06 cells/dish (6 cm diameter). After 4 days of cultivation, at the time of experiments, these cultures were practically free of non-neuronal cells ~. Glial cultures were obtained from 14-day-old embryonic forebrains after dissociation. Cells were seeded into Petri dishes (3-4 × 106 cells) and culti-

vated in the above medium for 11 days, until experiments were F:rformed. The neuronal contamination of these cultures was negligible is

Binding experiments [3H]Naloxone binding to intact cultures was measured according to a procedure described previously for fl-adrenergic receptors 17. Briefly, the cultures were washed with Tyrode's solution and preincubated in 1 ml serum-free Eagle MEM for 20 min at 37 °C in humidified air flow, containing 5% CO_,. The binding reaction was started by adding the radioligand [3H]naloxone into the medium of the cultures at 0.2-1.2 or 1.4 nM concentrations. Cells were incubated under the above conditions for 1 min in equilibrium experiments and I0 s to 15 min in association experiments. Non-specific binding was measured in the simultaneous presence of I !~M naloxone hydrochloride. Competition of unlabelled naloxone for specific binding sites was measured on glial cells at fixed 0.3, 0.45 or 0.6 nM [3H]naloxone and at increasing concentrations of naloxone hydrochloride (10 -~! to 10-5 M). Competition of the opioid drugs, morphine, EKC and DALA, was measured on both gliai and neuronal cultures. The fixed concentration of [3H]naloxone was 0.7 nM at glial and 0.8 nM at neuronal cells, while the concentration of unlabeled drugs increased in the range of l0 TM to 1(1-5M. Competitors were added simultaneously with the labelled ligand and were incubated with the cells for I rain under the conditions outlined above. The binding experiments were terminated by rapid and intensive washing of the cultures with physiological saline at room temperature. The bound radioactivity was quantitatively transferred to Bray's solution treatir,,g the wet cell layer with 2 x 500 ~! methanol. and measured in a liquid scintillation counter. Specific binding was calculated as the difference between total and non-specific binding. The non-specific binding amounted to 30% of the total in the case of neurons, but around 60% or more in glials. If glial cells were assayed in the presence of 1 ltM dextrorphan tartrate, this value decreased to 50% or less. Dextrorphan did not influence the character of binding and had no measurable effect on the binding of neuronal cells. In each experimental arrangement 2-3 indepen-

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RESULTS

[3H]Naloxone specific binding was measured in homogenous populations of glial and neuronal cells. As Fig. 1 shows, the association of [3H]naloxone was rapid, reaching equilibrium around 1 rain if measured at a 0.7 nM labelled iigand concentration. The equilibrium [3H]naloxone binding followed sigmoidal saturation kinetics in the range of 0.2-1.2 nM [3H]naloxone (Fig. 2A and B) in both cell types. The binding parameters have been estimated by

TABLE I

Parameters of [¢H]naloxone equilibrium specific binding Binding experiments were performed on intact glial and neuronal cells in culture. Parameters were obtained bv computer fitting of the Hill equation to the data presented on Fig. 2A and B. r = residual variance.

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44 computer fitting of the Hill equation and are given in Table I. The analysis of binding data revealed the apparent positive cooperativity of the [3H]naloxone binding, characterized by a Hill coefficient of n = 2.61 in glial and n = 2.04 in neuronal cells.The apparent equilibrium dissociation constants were calculated as K,t = 0.51 nM and 0.63 nM for glial and neuronal cells, respectively. The maximal binding capacities are given as specifically bound radioactivity of 106 cells (Bmax) and as the average number of binding sites per cell (S); however, this latter implie~ the assumption that all the cells have the same amount of receptors, which is not possible. These values are essentially different in the two cell types. The neuronal cells bound nearly 4 times more [3H]naloxone specifically than glial cells. The Bma x values are 4.9 and 1.1 fmol/106 cells (approx. 3000 and 700 bound molecules/cell), respectively. The inhibition of specific [SH]naloxone binding by unlabelled naloxone was measured on glial cells, at 0.3, 0.45 and 0.6 nM concentrations of labelled ligand, which corresponds to about 20%, 40% and 65% receptor occupancy. The results are given in Fig. 3. Analysing the process at lower receptor occupancies (20% and 40%), the unlabelled naloxone provoked maximum-shape competition curves, while at 65% occupancy ([3H]naloxone concentration higher than the apparent Kd) a curve, descending

with increasing naloxone concentrations was measured. These results provide further support 7 for the apparent positive cooperativity of ligand-binding of glial ceils. Competition of different unlabelled opioid ligands for [3H]naloxone specific binding sites was analysed in glial and neuronal cells, at 0.7 and 0.8 nM labelled ligand concentrations, respectively, corresponding to about 75% and 65% receptor occupancy values. Experiments are demonstrated in Fig. 4. Opioid agonists, EKC, morphine and DALA, given at 10TM to 10-5 M concentrations resulted in biphasic competition curves, similar in shape for both neurons and glial cells. The theoretical analysis of these curves, aiming at the estimation of apparent dissociation constants of competitors is in progress at present. The order of potency of agonists in inhibition of [3H]nalox-

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NALOXONECONCENTRATION(H) Fig. 3. Inhibition of specific [3H]naloxone binding of glial cells by unlabeiled m, loxone, measured at 0.3 nM ( A . A), 0.45 nM ((3------C)) and 0.6 nM (O------O) labelled ligand concentrations, which corresponds to 20%, 40% and 65% relative occupancy. It:tact cells were incubated in the simultaneous oresenee nf Inh~l,~ d and ,,,.t.~...:...~ naloxone for 1 tnin. Details are given in Materials and Methods. 100% control values are 0.8, 1.6 and 2.5 ~moi/dish (3.5 x 106 cells), respectively. Average of two experiments. •

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10-11 10-10 10-9 10-8 10-/ 10-6 10-5 COHPETITOR CONCENTRATION(H) Fig. 4. Competition of EKC (O------O), morphine (A &) and DALA (O ©) for [3H]naloxone binding sites of intact glial cells (A) and neurons (B) of chick embryonic forebrain. Cultures were incubated for 1 min in the simultaneous presence of competitors and 0.7 nM (glial cells) or 0.8 nM (neurons) [3H]naloxone, For details see Materials and Methods. 100% control vaiues are 3.2 fmoi/dish (4 x i0 ° cells) for giiai cultures and 3.06 fmol/dish (106 cells) for neuronal cultures, corresponding to about 75% and 65% fractional occupancy. Average of two determinations.

45 one specific binding proved to be: EKC > morphine > DALA, in both cell types studied. DISCUSSION

Endogenous opiates are supposed to act as modulators in different cellular functions 22"24"33"34in many cases through the adenylate cyclase system 3"5"19"22"24. According to data summarized by Osborne et al. 2°, a possible target of endogenous opioid and other peptides may be the glial cells. Some authors ~'22"24premised the presence of opioid receptors on glial cells; their measurement by radioligand binding, however, was unsuccessful l' 12. Here we provide evidence on the opioid receptors of normal, intact gliai cells of chick embryonic brain by [3H]naloxone binding. The experiments were completed with the measurement of neuronal cells of the same origin. The technique, applied here for radioligand binding, was worked out previously for the study of fl-adrenergic receptors of intact brain cells 17.18. Our results demonstrate that the opioid receptors of gliai cells are basically similar to that of neuronal cells. The apparent Kd values are in the range of 0.5-0.6 nM, which correspond well with the high affinity opioid receptor population measured by several authors in brain fragments (refs. 10, 15, 21.31 as examples), neuronal membranes 12 or intact neuroblastoma x glioma hybrid cells 12. The binding capacities of the two cell types are essentially different. We estimated about 700 opioid binding sites on glial cells and nearly 5 times more, i.e. 3000 on neurons. The relatively low receptor number of glial cells, which was already supposed by Rougon et al. 24, and the higher non-specific binding of the glial membrane may explain among other possibilities, why difficulties arose in the measurement of this receptor. An apparent positive cooperativity characterizes the [3H]naloxone equilibrium binding of both neuronal and glial cells. The Hill coefficients are n > 1 (2.04 and 2.61, respectively). This character is supported by the results of cold naloxone competition experiments (see Fig. 3). Biphasic curves were measured at receptor occupancy values less than 50%, with highest peak at lowest occupancy 7. The positive cooperativity of the opioid receptor li-

gand-binding was first demonstrated by Davies et al. 7 in chick brain slices, using [3H]morphine as a iigand. The authors also reported the lack of this cooperativity in homogenized brain fragments. Similar cooperative binding was presented by Scatchard analysis of [3H]naltrexone binding of rat cerebral membranes "~. The app,.rent cooperativity may have originated from the multiplicity of the opioid receptors, as Davies et al. 7 suggested. Furthermore allosteric interactions or conformational changes of the opioid receptors 8 as well as the multiple states of the opioid binding sites 6'13may be the reason why apparent cooperativity of the ligand binding can be measured. It is of interest, that the apparent positive cooperativity of the opioid receptors was reflected also in the competition pattern of the different opioid iigands. The ligands tested in this experiment were of agonist type, with relative selectivity for r (EKC),/~ (morphine) or 6 (DALA) opioid receptors. The agonists caused a characteristic maximum in the [3H]naloxone relative occupancy curve. The theoretical evaluation of these results is at present under study. We aim to establish a method for the estimation of the apparent dissociation constant for competitors with much higher accuracy as can be estimated from the IC50 values. Comparing neuronal and glial cells in this experiment, the order of potency of competitors was found to be: EKC>morphine>DALA, in both cell types. Taking into account that the cells in our cultures are not fully differentiated, these results seem to be in agreement with the data that supports the appearence of l~ and K opioid binding sites preceding that of b sites in the developing brain of rat, mouse and sheep 9"16,28. Our results clearly demonstrate opioid receptors on cultured glial cells.The characteristics of these receptors are basically similar to that of the neuronal cells; their number, however, is much lower. As far as the physiological significance of glial opioid receptors is concerned, we suppose that they are involved in neuronal-glial communication. This view is based on the finding, that neurons of different transmitter types produce also neuropeptides ~4'2~ and a part of these nerve terminals have been found apposed to glial cells (astrocytes) 2. Recently Osborne ~ summarized data which suggest that glial cells are one possible target of neuropeptides, and in this manner neurons may modulate glial functions.

46 The apparent positive coooerativity of the opioid receptors presented here, demonstrates an extremely sensitive system at low ligand concentrations. Studying the fl-adrenergic receptors of these cells, we have already discussed rl"18 the significance of this mechanism in the perception of weak, modulatory stimuli, operating also in the glial-neuronal communication. The confirmation of this possibility for the

opioid receptors, however, necessitates further investigations.

REFERENCES

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ACKNOWLEDGEMENTS

The authors are indebted to Dr. Maria Wollemann for valuable discussions and to Miss Zsuzsanna Magyar for excellent technical assistance.

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